Local PI(4,5)P_2 synthesis by septin-associated PIPKIγ isoforms controls centralspindlin association with the midbody during cytokinesis

Domain: neuroscience
Published: 2026-02-07
Editorial outcome: major_revision

Abstract
Cytokinesis critically depends on phosphatidylinositol 4,5-bisphosphate [PI(4,5)P ]. Synthesis of PI(4,5)P is crucial for several stages of cytokinesis, including actomyosin ring assembly and constriction, membrane tethering of spindle microtubules, and midbody organization. How these activities of PI(4,5)P are spatiotemporally controlled is unknown. Here we unravel a crucial function for local PI(4,5)P synthesis at the ingressed cleavage furrow by septin-binding isoforms of PIPKIγ to control midbody formation. We demonstrate that loss of PIPKIγ isoforms perturbs cytokinesis by impairing septin association with microtubules, and anillin and septin deposition at the intercellular bridge and at the midbody. This mechanism requires the ability of PIPKIγ isoforms to synthesize PI(4,5)P and to associate with septins. Septins and PIPKIγ further synergize to promote centralspindlin recruitment to the midbody. Our findings establish septin-associated PIPKIγ isoforms as spatiotemporal controllers of midbody organization during cytokinesis that act through generating a local pool of PI 4,5P at the ingressed cleavage furrow.

Introduction
Cytokinesis is the final step of cell division that ultimately partitions the cytoplasmic content of the mother cell into two daughter cells. Failures in cytokinesis can cause tetraploidy, centrosome amplification, and chromosomal instability, and thereby promote tumorigenesis1. Cytokinesis starts with the assembly of a contractile actomyosin ring at the equatorial plane of the mother cell that drives the formation of the cleavage furrow2,3. The cleavage furrow ingresses symmetrically, until the daughter cells are connected only by a thin intercellular bridge (ICB) formed around a tight bundle of central spindle microtubules4, the cytokinetic bridge. As the ICB elongates, a dense structure assembles and matures in its center, the midbody, an organelle of complex and unique protein and lipid composition5. The midbody serves several important functions, including tethering of spindle microtubules to the plasma membrane and the orchestration of the abscission machinery6.

Cytokinesis is accompanied by drastic morphological changes of the mother cell, which rely on continuous remodeling and reorganization of the cytoskeleton. These events are orchestrated by phosphoinositides7, in particular by the plasma membrane-enriched signaling lipid phosphatidylinositol 4,5-bisphosphate [PI(4,5)P]8. PI(4,5)P serves as a docking site for several components of the cytokinetic machinery9. It accumulates early at the newly forming cleavage furrow, where it anchors the contractile machinery to the cell equator to ensure symmetric ingression of the furrow10. This process is coordinated by anillin, a multidomain protein that scaffolds actin, myosin, septins, and the central spindle11 and via its PI(4,5)P -binding pleckstrin homology (PH) domain attaches this scaffold to the plasma membrane12,13. Stable anchorage of anillin at the cleavage furrow also depends on RhoA13, which is activated by microtubule-associated centralspindlin through Ect-2 at metaphase14, and stabilized at the cortical membrane in an anillin- and PI(4,5)P -dependent fashion15.

As the contractile ring closes, PI(4,5)P further concentrates along the cleavage furrow, and later at the ICB to support bridge stabilization10,16. In addition to PI(4,5)P, ICB stability is also fostered by septins, a family of filament-forming, GTP-binding proteins17,18 that are recruited to the cleavage furrow through direct interactions with anillin10,12 and with PI(4,5)P . Septins are required for symmetric cleavage furrow ingression, and, by an unknown mechanism, promote stable anchorage of the ingressed furrow at the cytokinetic bridge to prevent furrow regression upon completion of contraction19,20. Septin filaments associate with membranes enriched in PI(4,5)P in vitro and in living cells21–23, and several lines of evidence indicate that PI(4,5)P is required for the localization of septins at the ICB (reviewed in ref. 24).

At later stages of cytokinesis, when the midbody has formed, PI(4,5)P tethers spindle microtubules to the plasma membrane through centralspindlin25, a constitutive heterotetramer of MKLP1 (also known as KIF23) and MgcRacGAP (also known as RacGAP1)26. Whereas MKLP1 promotes bundling of antiparallel spindle microtubules at the midbody, MgcRacGAP locally controls the activities of Rho GTPases27 and, through an atypical C1 domain, associates with plasma membrane PI(4,5)P and phosphatidylinositol 4-phosphate [PI(4)P]25. Coincidentally, PI(4,5)P facilitates exocyst docking to the plasma membrane of the ICB, and thereby supports the abscission process28.

An unsolved key conceptual problem is how the various distinct roles of the highly diffusible signaling lipid PI(4,5)P during multiple stages of cytokinesis are controlled in space and time. In particular, it is unknown how PI(4,5)P synthesis is mechanically coupled to cytokinetic progression and midbody formation. One possibility is that distinct isoforms of type I PI(4)P 5-kinases, the main PI(4,5)P -synthesizing enzymes in mammalian cells, operate at distinct stages of cytokinesis and at defined sites. Type I PI(4)P 5-kinases are encoded by PIPKIα, Iβ, and Iγ genes, with several splice isoforms being known for PIPKIγ29. All isozymes are widely expressed in different tissues and cell types, but exhibit unique subcellular distributions, and trigger the formation of functionally distinct pools of PI(4,5)P . The mechanisms underlying the specific subcellular distribution of select isozymes remain incompletely understood. In the case of PIPKIγ, short splice inserts target kinase activity to distinct locations for defined subcellular events. For example, isoform i2 contains a short peptide that associates with AP-2 and talin to promote kinase recruitment to sites of endocytosis30–32 and to focal adhesions33–35, respectively. Isoform i5 localizes to endosomes36 and autophagosomes37, due to its interactions with SNX5 and Atg14.

PIPKIγ recruitment factors are often also PI(4,5)P effectors, and, thus, considered part of a positive feedback loop that enhances PI(4,5)P synthesis in their immediate proximity to reinforce their own membrane association38. It is likely that similar mechanisms underlie the recruitment of cytokinetic proteins, but up to now, no interactions with the PI(4,5)P-synthesizing machinery have been revealed.

Here, we close this fundamental knowledge gap regarding the mechanism of cell division by unraveling a function of PIPKIγ during ICB maturation and late midbody formation. We demonstrate that splice isoforms i3 and i5 associate with septins, and that this interaction is essential for the retention of anillin and septins at the ICB. We further show that the septin-dependent recruitment of PIPKIγ to the ICB is required for efficient targeting of centralspindlin to the midbody, and for the translocation of septins onto microtubules during late stages of cytokinesis. These findings establish septin-associated PIPKIγ isoforms as spatiotemporal controllers of midbody organization during cytokinesis that act through generating a local pool of PI 4,5P at the ingressed cleavage furrow. Given the signaling functions of postmitotic midbodies in regulating cell polarity and proliferation39, these findings provide an avenue for future studies on how the septin/PIPKIγ-dependent assembly of the midbody affects its downstream signaling, also beyond cytokinesis.

Results
PIPKIγ exhibits a unique function at late stages of cytokinesis
The pivotal role of PI(4,5)P during mitosis is well established, but little is known about the specific enzymes mediating its synthesis during cytokinesis. In mammalian cells, PI(4,5)P is mainly synthesized by type I PIPK enzymes, named PIPKIα, Iβ, and Iγ. To identify the individual contributions of distinct type I PIPK enzymes, we depleted PIPKIα, Iβ, and Iγ isozymes in HeLa cells and subsequently analyzed the cells for cytokinetic defects. We immunostained cells for acetylated tubulin, a marker of spindle microtubules before furrow ingression, and of microtubules within the ICB from telophase until abscission (Fig. 1a). Approximately 2% of the cells displayed a mitotic spindle under control conditions (Fig. 1b; Supplementary Fig. 1a, b). Depletion of PIPKIβ, but not of PIPKIα, significantly increased this fraction, in line with previous reports16. Loss of PIPKIγ also raised the fraction of cells exhibiting an acetylated tubulin spindle, i.e., cells in meta- and anaphase. This was surprising as this isozyme is believed to contribute only a minor fraction of total PI(4,5)P in non-neuronal cells29. More importantly, depletion of PIPKIγ, but not of Iα or Iβ, doubled the fraction of cells exhibiting an acetylated tubulin bridge, indicative of stalling in telophase (Fig. 1c). The resulting mitotic arrest led to an approximately threefold increase in the fraction of multinucleated cells (Supplementary Fig. 1c, d).

Our analyses also revealed other abnormalities in PIPKI-depleted cells, such as multipolar spindles or bridges. Loss of PIPKIβ or Iγ caused a significant increase in the fraction of cells that displayed a multipolar spindle (Fig. 1d; Supplementary Fig. 1e), while solely the depletion of PIPKIγ resulted in a significant increase in cells with multipolar bridges (Fig. 1e; Supplementary Fig. 1e). Depletion of neither type I PIPK isozyme significantly changed the overall PI(4,5)P levels at the plasma membrane (Supplementary Fig. 1f, g). This was expected, as despite their unique tissue distribution and distinct subcellular localizations, PIPKIα, Iβ, and Iγ are able to largely compensate for each other40 with respect to overall synthesis of PI(4,5)P .

These data reveal that while PIPKIβ and Iγ have synergistic roles during furrow ingression, PIPKIγ exhibits a hitherto unknown, unique function at late stages of cytokinesis, when the ICB is formed.

PIPKIγ is required to concentrate the PI(4,5)P effectors anillin and septins during ICB maturation
Given the unique defects observed upon depletion of PIPKIγ at late stages of cytokinesis, we analyzed the subcellular distribution of PI(4,5)P effectors such as anillin in HeLa cells, that were synchronized by a thymidine/nocodazole block and fixed at a stage when they exhibit a mature ICB. Anillin bears a PI(4,5)P -binding PH domain in its C-terminus11,12 that facilitates its recruitment to the equatorial plane of dividing cells. Upon closure of the contractile ring, anillin is found concentrated at the midbody, where it acts in concert with septins to anchor the midbody to the membrane41, and—prior to abscission—follows septins to the midbody flanks42. Control cells or cells depleted of PIPKIα or Iβ displayed a compact organization of anillin at the ICB as visualized by co-staining for acetylated tubulin (Fig. 1f, g). In contrast, PIPKIγ knockdown cells lacked a compact organization of anillin (Fig. 1g), which instead was found scattered along the bridge. Additional clusters of anillin were detected in areas adjacent to the bridge, which were rarely observed under control conditions.

Through its C-terminal PH domain, anillin associates with septins43, a family of guanine nucleotide-binding hetero-oligomerizing proteins, in a RhoA-dependent manner44. An additional septin binding interaction surface is provided by the anillin N-terminus, which indirectly associates with SEPT9-enriched septins through CIN8545. PI(4,5)P promotes septin association with membranes in living cells and in vitro21–23. Although largely dispensable during furrowing in mammalian cells, septins exert pivotal functions once the ICB is established and extends42 by compartmentalizing select cytokinetic proteins at the midbody19 and by scaffolding the assembly of the abscission machinery42,46. To analyze how individual PIPKI isozymes affect septin localization during cytokinesis, we assessed the subcellular distribution of SEPT2, a highly abundant septin isoform present in hexa- and octamers. In the vast majority of control cells, or in cells depleted of PIPKIα or PIPKIβ, SEPT2 was found to be enriched at the ICB (Fig. 1h) with about 5% of total cellular SEPT2 residing on the acetylated tubulin bridge (Fig. 1i). Depletion of PIPKIγ led to a marked loss of SEPT2 from ICB microtubules. Instead, SEPT2 remained associated with the plasma membrane of the newly forming daughter cells at sites adjacent to the bridge membrane (Fig. 1h, i).

These findings show that PIPKIγ spatiotemporally controls the organization of anillin and septins at the ICB.

Distinct splice variants of PIPKIγ associate with septins and colocalize with septin filaments
The carboxyterminal region of human PIPKIγ undergoes alternative splicing, which gives rise to five isoforms termed PIPKIγ-i1-i5 (Fig. 2a). The distinct splice inserts promote PIPKIγ association with select binding partners at specific subcellular localizations29 and this mechanism is thought to direct the generation of PI(4,5)P at defined sites. To identify novel binding partners of these splice variants that might connect PIPKIγ to the cytokinetic machinery, we performed affinity purification experiments. Surprisingly, we discovered that glutathione-S-transferase (GST)-fused carboxyterminal tails of both PIPKIγ-i3 and -i5 efficiently retained a variety of septin isoforms (Fig. 2b), indicative of a specific interaction of PIPKIγ-i3 and -i5 with septin oligomers. PIPKIγ-i2 and i3 efficiently associated with the focal adhesion protein talin from mouse brain lysates (Fig. 2b) as expected based on the presence of a talin binding-specific splice insert and on prior data33–35 (Fig. 2a). Moreover, we found mCherry-PIPKIγ-i5 to co-immunoprecipitate myc-tagged SEPT7 or SEPT9 from HEK-293T cell lysates, but not endogenous talin (Supplementary Fig. 2a, b). PIPKIγ also co-purified with endogenous septins from extracts of mitotic HeLa cells (Fig. 2c). These data indicate that PIPKIγ isoforms undergo complex formation with septins in living cells. We then performed alanine scanning mutagenesis along the i3/i5-specific splice insert and identified two neighboring residues (Y646 and W647) that are critically involved in septin binding: A point mutant variant of the PIPKIγ-i5 tail, i.e., Y646A/W647A, hereafter referred to as PIPKIγ-i5ΔSB (“deficient in septin binding”), failed to affinity-purify septins (Fig. 2b).

Next, we investigated a potential colocalization of PIPKIγ splice variants with septins. To this end, we transfected HeLa cells with HA-tagged versions of PIPKIγ. Notably, when expressed at low levels, HA-PIPKIγ-i3 and -i5 were organized in filaments that overlapped with endogenous septin fibers visualized by immunostaining of SEPT6 (Supplementary Fig. 2c). No such colocalization was observed for PIPKIγ-i1 or -i2, or for septin-binding-deficient mutant PIPKIγ-i5ΔSB. We corroborated these findings in genome-edited Norwegian Rat Kidney fibroblasts (NRK49F) that express SEPT2-eGFP under its endogenous promoter47. In this cell line, septins display a characteristic prevalent association with actin filaments (Fig. 2d). mCherry-PIPKIγ-i3 and -i5, but not septin-binding-deficient mutant PIPKIγ-i5ΔSB, colocalized with endogenous SEPT2 (Fig. 2e). PIPKIγ-i5 was also detected on intracellular organelles, in agreement with its known association with late endosomes and autophagosomes36,37. In contrast, mCherry-PIPKIγ-i1, i2, or i4, i.e., isoforms that do not bind to septins, were not colocalized with septin filaments. PIPKIγ-i1 was uniformly distributed across the plasma membrane, i2 additionally localized to focal adhesions33,34, and i4 was detected in nuclear foci, presumably representing speckles48.

F-actin is known to serve as a template for the assembly of septins into filaments49. Moreover, it has been shown that treatment with cytochalasin D releases septins from actin filaments and induces their reorganization into rings49. To rule out the possibility that the colocalization of septins with mCherry-PIPKIγ-i5 is bridged by actin, we treated NRK49F cells with cytochalasin D. Application of the drug triggered a rapid collapse of SEPT2-eGFP-containing filaments into rings, as seen before20,49. These rings retained their colocalization with mCherry-PIPKIγ-i5, but lost their association with F-actin (Fig. 2f, g; Supplementary Fig. 2d). Similar observations were made for catalytically inactive mutant mCherry-PIPKIγ-i5 (K188A).

Together, these findings demonstrate that PIPKIγ splice isoforms i3 and i5 associate with septins in living cells via a mechanism that is independent of lipid kinase activity or the association of septins with F-actin. Given the pivotal role of septins during cell division, these data suggest that the functions of PIPKIγ during late stages of cytokinesis may be exerted by its i3 and i5 splice variants.

Spatiotemporally controlled PI(4,5)P synthesis at the midbody by septin-associated PIPKIγ-i3/i5 promotes anillin recruitment
To directly test the role of PIPKIγ-i3/i5 in cytokinesis, we analyzed the subcellular distributions of PIPKIγ-i3/i5, anillin, and septins at different stages of mitosis. At anaphase, PIPKIγ-i5 was homogenously distributed at the plasma membrane of HeLa cells stably expressing mCherry-PIPKIγ-i5 (Fig. 3a). SEPT2 was enriched at the equatorial plane and at the poles of the mother cell, whereas anillin was found exclusively at the equatorial plane. During the transition to telophase, mCherry-PIPKIγ-i5 became concentrated at the cleavage furrow, where it aligned with SEPT2 and anillin (Fig. 3a). Upon completion of furrowing, mCherry-PIPKIγ-i5 localized to the ICB, where it outlined the midbody. At this stage, anillin accumulated at the midbody while SEPT2 started to translocate onto microtubules. At telophase, the distribution of mCherry-PIPKIγ-i3 at the midbody was indistinguishable from that of PIPKIγ-i5 (Supplementary Fig. 3a). This spatiotemporal choreography is consistent with a model in which PIPKIγ-i3/5 executes a local function at the ICB (e.g., synthesis of PI(4,5)P ) to promote the ingression of the cleavage furrow, possibly by enabling the recruitment of anillin and other factors.

To investigate whether the disorganization of anillin and septins at the ICB of PIPKIγ-depleted cells (Fig. 1f–i) reflects a selective loss-of-function of septin-binding isoforms of PIPKIγ, we depleted PIPKIγ-i3/i5 with an siRNA that targets the common splice insert36. This treatment reduced mRNA levels of the two cognate splice variants by about 85%, but did not affect the expression levels of other isoforms of PIPKIγ (Supplementary Fig. 3b, c). Select depletion of PIPKIγ-i3/i5 increased multinucleation to a similar degree as seen for the general depletion of all PIPKIγ transcripts (Supplementary Fig. 3d). This was accompanied by a dramatic scattering of anillin from the center of the ICB to its periphery (Fig. 3b, c). We also found that depletion of PIPKIγ-i5 alone caused a 1.9-fold increase in multinucleation, i.e., a phenotype milder than that elicited by combined loss of both PIPKIγ-i3 and -i5 (3.3-fold increase in multinucleation). This indicates that PIPKIγ-i3 is indeed expressed in HeLa cells, in agreement with an earlier report50. Moreover, these data suggest that PIPKIγ-i3 and PIPKIγ-i5 likely exhibit overlapping functions in cytokinesis in HeLa cells. Importantly, multinucleation as induced by depletion of PIPKIγ-i3/i5 was efficiently rescued in cells stably expressing siRNA-resistant, mCherry-tagged PIPKIγ-i5, but not in cells expressing mCherry (Supplementary Fig. 3e). Loss of PIPKIγ-i3/i5 did not affect the recruitment of anillin or septins to the cleavage furrow at earlier stages of cytokinesis (Supplementary Fig. 3f), emphasizing the predominant role of septin binding kinase isoforms during maturation of the ICB. Consistently, when monitoring microtubule dynamics in living cells upon application of SiR-tubulin we failed to observe significant changes in furrow ingression time upon depletion of PIPKIγ-i3/i5 (Supplementary Fig. 3g) Active myosin at the ingressed furrow, as evaluated by immunostainings of phospho-myosin light chain, was also unaltered (Supplementary Fig. 3i). By contrast, we observed a marked delay in the timing of microtubule severing (Supplementary Fig. 3h).

We hypothesized that anillin scattering from the ICB in cells depleted of PIPKIγ-i3/i5 might be a consequence of impaired local synthesis of PI(4,5)P. To test this, we first analyzed the impact of loss of PIPKIγ-i3/i5 on the levels and localization of PI(4,5)P at the midbody of synchronized cells. In control cells PI(4,5)P was found at the outlines of the ICB including regions adjacent to the midbody marked by citron kinase (Fig. 3d). Loss of PIPKIγ-i3/i5 resulted in a significant decrease in PI(4,5)P at the midbody (Fig. 3d, e). Depletion of OCRL (oculocerebrorenal syndrome of Lowe protein), a PI(4,5)P 5-phosphatase that is delivered to the ICB by Rab35 prior to abscission to locally erase PI(4,5)P51, led to a more than two-fold increase in PI(4,5)P levels along the ICB and at the midbody (Fig. 3d, e). The distribution of citron kinase remained unaffected by either knockdown condition (Fig. 3d, f). Next, we conducted genetic epistasis experiments to test if co-depletion of PIPKIγ-i3/i5 and the PI(4,5)P 5-phosphatase OCRL can rescue defective anillin recruitment to the ICB. Knockdown of OCRL alone modestly increased the fraction of cells with anillin at their ICBs (Fig. 3g, h; Supplementary Fig. 3j). Co-depletion of OCRL together with PIPKIγ-i3/i5 rescued the mislocalization of anillin observed upon loss of PIPKIγ-i3/i5 alone. Third, we conducted rescue experiments using wild-type or catalytically inactive mutant PIPKIγ-i5, unable to synthesize PI(4,5)P. We observed that anillin concentration at the ICB was fully restored by reexpression of mCherry-tagged, siRNA-resistant wild-type PIPKIγ-i3 or i5 (consistent with their overlapping functions, see above), but not by inactive mutant PIPKIγ-i5 (Fig. 3i, Supplementary Fig. 3k). Intriguingly, the septin-binding-defective mutant PIPKIγ-i5ΔSB also failed to rescue anillin dispersion. Failure to rescue anillin dispersion was associated with mistargeting of mutant PIPKIγ-i5: Whereas wildtype mCherry-PIPKIγ-i5 was found to be enriched at the ICB in close proximity to endogenous anillin (Fig. 3a, Supplementary Fig. 3k), mCherry-PIPKIγ-i5 K188A, or ΔSB, were spread along the plasma membrane adjacent to the bridge, resembling the dispersed distribution of anillin (Supplementary Fig. 3k).

Taken together, these data suggest a model according to which septins anchor cognate PIPKIγ variants i3 and i5 at the midbody to produce a local pool of PI(4,5)P that stably anchors anillin.

PIPKIγ-i3/i5 is required for the organization of septins and microtubules at the ICB
Given the striking impact of septin-associated PIPKIγ-i3/i5 on midbody PI(4,5)P levels and on anillin localization, we assessed their role in the dynamic reorganization of the septin cytoskeleton during cytokinesis. To this end, we generated a genome-edited cell line expressing eGFP-SEPT6 from its endogenous promoter. We obtained several heterozygous knock-in clones, in all of which endogenous SEPT6 and eGFP-SEPT6 protein levels decreased upon treatment with SEPT6-targeting siRNA (Supplementary Fig. 4a). Furthermore, eGFP-SEPT6 was successfully incorporated into septin filaments (Supplementary Fig. 4b) and displayed a high degree of colocalization with endogenous septin paralogs (Supplementary Fig. 4c), indicating that endogenous eGFP tagging does not interfere with SEPT6 function. Next, we monitored the subcellular distribution of eGFP-SEPT6 during cytokinesis (Supplementary Movie 1; representative frames reported in Supplementary Fig. 4d). Under control conditions, eGFP-SEPT6 first accumulated at the cleavage furrow (white asterisk in Supplementary Fig. 4d). About 30 min after completion of furrow ingression the ICB appeared. At the same time, eGFP-SEPT6 started to relocate from the cell cortex onto putative microtubules within the ICB and remained there during bridge elongation until abscission. After abscission the daughter cells displayed perinuclear, sinuous septin fibers that likely derived from the ICB (white arrowheads). In cells depleted of PIPKIγ-i3/i5 eGFP-SEPT6 retained its initial localization at the cleavage furrow but failed to accumulate at the ICB and to translocate onto microtubules. Moreover, the resulting daughter cells lacked the prominent septin fibers observed under control conditions. This defect persisted throughout interphase (Supplementary Movie 2; representative frames reported in Supplementary Fig. 4d). In addition, we observed that about 29.2% (±8.4% SEM, n=3) of kinase-depleted cells died during cell division, compared to 6.6% (±4.3% SEM, n=3) of control cells. As a result, only about 60.6% (±3.5% SEM, n=3) of knockdown cells underwent successful division, compared to 82.7% (±6.5% SEM, n=3) of control cells.

Septins are well known to associate with microtubules to regulate their stability and dynamics18. Given the dramatic impact of PIPKIγ on septin distribution, we investigated the organization of ICB microtubules in detail. Depletion of PIPKIγ-i3/i5-induced scattering of septin filaments away from the acetylated tubulin bridge (Fig. 4a, b), similar to cells lacking all PIPKIγ isoforms (compare Fig. 1h). Furthermore, microtubule stability assessed by acetylated tubulin was impaired (Fig. 4a, c). Loss of PIPKIγ-i3/i5 also significantly reduced the length of the acetylated tubulin bridge (Fig. 4d) and microtubule bundling along the ICB (Fig. 4a, e). Consistent with the latter observation, ICBs of kinase-depleted cells accumulated significantly lower levels of the microtubule bundling factor PRC1 (Supplementary Fig. 4e, f). The defective septin organization at the ICB (Fig. 4f, Supplementary Fig. 4g), the reduced length of the acetylated tubulin bridge (Fig. 4g, Supplementary Fig. 4g), and the defective microtubule bundling (Fig. 4h, Supplementary Fig. 4g) were rescued by expression of wild-type mCherry-PIPKIγ-i3 or -i5, but not of -i5 mutants lacking catalytic activity or the ability to associate with septins.

In summary, these data demonstrate that PIPKIγ-i3/i5 isoforms are required for the organization of septins and microtubules at the ICB via a mechanism that depends on their ability to locally synthesize PI(4,5)P and to bind to septins.

Septin-dependent recruitment of PIPKIγ is essential to maintain centralspindlin at the midbody
The observed impact of PIPKIγ-i3/i5 depletion on microtubule organization and stability prompted us to analyze the effect of PIPKIγ-i3/i5 loss on midbody-associated microtubule-binding proteins, in particular on centralspindlin. The centralspindlin protein complex is composed of MgcRacGAP and mitotic kinesin-like protein 1 (MKLP1) and tethers midbody microtubules to the plasma membrane26. MgcRacGAP comprises an atypical C1-domain with affinity for PI(4,5)P25. Given the impact of PIPKIγ-i3/i5 depletion on septin and microtubule organization during cytokinesis, we hypothesized that the septin-dependent recruitment of PIPKIγ is required for the stable association of centralspindlin with the cell cortex. To test this hypothesis, we assessed the localization of centralspindlin at late stages of cytokinesis. As expected, under control conditions, MKLP1 colocalized with the midbody ring visualized by immunostaining for citron kinase (Fig. 5a). Notably, knockdown of PIPKIγ-i3/i5 had no impact on the distribution of citron kinase or on its abundance at the midbody (Fig. 5a, b). Loss of PIPKIγ-i3/i5, thus, does not generally affect midbody composition or positioning. In contrast, depletion of PIPKIγ-i3/i5 significantly decreased MKLP1 levels at the midbody ring (Fig. 5a, c). This defect was rescued by expression of wild-type mCherry-PIPKIγ-i5, but not catalytically inactive or septin-binding-deficient kinase mutants (Fig. 5d, Supplementary Fig. 5a). Depletion of SEPT2 impaired the deposition of centralspindlin at the midbody to an extent similar to that observed upon loss of PIPKIγ-i3/i5 (Fig. 5e, f). These results are consistent with a primary role of septins in guiding PIPKIγ to the ICB.

Next, we aimed at gaining insight into the spatial organization of septins, microtubules, and centralspindlin at the midbody. The detection of midbody proteins by conventional fluorescence microscopy is complicated by the dense packing of individual components in this organelle, which allows only limited access for antibodies. To solve this problem, we used ultrastructure expansion microscopy (U-ExM)52, for which the specimen is physically expanded prior to antibody labeling53. At cytokinesis control cells exhibited bundles of antiparallel microtubules along the ICB that overlapped at the midbody (Supplementary Fig. 5b). Septins were not found at the midbody itself, but formed two rings that aligned with two secondary ingression sites at its flanks (Supplementary Fig. 5b). Additionally, septins colocalized with microtubules that protruded away from the secondary ingression sites into the developing daughter cells. Upon depletion of PIPKIγ-i3/i5, septins underwent a dramatic redistribution: they were mostly dispersed from the ICB and only sparsely aligned with microtubules (Supplementary Fig. 5b). Instead of being organized in continuous straight fibers, they formed short rods and curly filaments in proximity to the ICB at the periphery of the daughter cells. We then assessed the distribution of centralspindlin by U-ExM. In control cells, centralspindlin localized to a confined region at the midbody where microtubules overlapped. In PIPKIγ-i3/i5-depleted cells, total centralspindlin levels at this locale were significantly reduced, akin to our findings in non-expanded specimens (Fig. 5g).

The PI(4,5)P -dependent recruitment of effector proteins to the plasma membrane is often fostered by complex formation between an effector protein and a PI(4,5)P-synthesizing enzyme to locally boost PI(4,5)P synthesis38. This raises the possibility that centralspindlin might interact with PIPKIγ. To test this, we screened for binding partners of septins and PIPKIγ in cytokinetic HeLa cells. We identified the centralspindlin component MKLP1 in immunoprecipitates of endogenous SEPT2 and of endogenous PIPKIγ (Fig. 5h, i). Citron kinase, a known interactor of centralspindlin54, was detected in both immunoprecipitates as well. Anillin efficiently copurified with septins, but not with PIPKIγ. Hence, the association of septins with PIPKIγ is not bridged by anillin. Furthermore, the microtubule bundling factor PRC1 reliably precipitated with SEPT2 and recruited septins onto hyper-bundled microtubules in transfected fibroblasts (Supplementary Fig. 5c), suggesting a role of the microtubule bundling machinery in septin recruitment onto microtubules.

Next, we investigated how protein levels of PIPKIγ might be affected by key partners identified in this study. Depletion of anillin or of MgcRacGAP led to significantly decreased levels of PIPKIγ (Supplementary Fig. 5d), whereas knockdown of SEPT7, a subunit required for maintaining septin cytoskeleton integrity, did not affect PIPKIγ levels. This indicates that anillin-41 and MgcRacGAP-25 mediated midbody tethering to the plasma membrane is important for PIPKIγ stability. Finally, we depleted anillin, McgRacGAP, or SEPT7 in stably transfected, synchronized HeLa cells to monitor the subcellular distribution of mCherry-tagged PIPKIγ-i5 (WT). In control cells, mCherry-PIPKIγ-i5 prominently outlined late-stage ICBs (Supplementary Fig. 5e), as expected. Cells depleted of anillin, MgcRacGAP, or SEPT7 showed severe mitotic defects, consistent with previous reports19,25,43, and only a few cytokinetic cells displayed an elongated acetylated tubulin bridge. Of note, however, in those cells, mCherry-PIPKIγ-i5 appeared largely excluded from ICBs (Supplementary Fig. 5e). We conclude that stable retention of septin-binding splice variants of PIPKIγ at the ICB requires concomitant presence of anillin, MgcRacGAP, and intact septin filaments.

Taken together, these findings demonstrate that the spatiotemporally controlled recruitment of septin-associated PIPKIγ isoforms to the ICB is essential to maintain centralspindlin at the midbody, via a mechanism that involves the local generation of PI(4,5)P , which in turn is required for microtubule organization at the ICB.

Methods
Plasmids and siRNAs
For mammalian expression constructs, the coding sequences (CDSs) of human PIPKIγ isoforms 1–5 (PIPKIγ-i1-i5)50, and of i5 mutants were inserted into pcDNA3.1(+)-based vectors, resulting in the expression of N-terminally mCherry- or HA-tagged proteins. The mutant PIPKIγ-i5 K188A carries the previously described mutation within the kinase core domain, which renders the kinase inactive (Krauss et al., 2006). The mutant deficient in septin binding, PIPKIγ-i5 ΔSB, was generated by mutating Y646 and W647 of human PIPKIγ-i5 into alanine by site-directed mutagenesis (Y646A/W647A). SiRNA-resistant PIPKIγ-i3, -i5, and -i5 mutants were created by introducing four silent mutations within the sequence targeted by the siRNA against PIPKIγ-i3/i5 as follows: 5′-CGACGGCAGATACTGGATT-3′. For viral constructs, the resulting CDSs were subsequently inserted into pLIB-CMV-mCherry-IRES-Puro. For bacterial expression constructs, the CDSs of the tail domains of PIPKIγ-i1-i5 and -i5 ΔSB (aa 451 to end) were inserted into pGEX-4T1, allowing for the expression of N-terminally tagged glutathione-S-transferase (GST) fusion proteins.

His -PRC1 was a gift from Stephen Royle (Addgene plasmid #69111; http://n2t.net/addgene:69111)69. The coding sequence was subcloned into a pcDNA3.1(+)-based vector, resulting in the expression of an N-terminally eGFP-tagged protein.

Most siRNAs used in this study were purchased from Sigma-Aldrich and had 3′-dTdT overhangs. The following siRNAs were used, targeting the human sequences: siOCRL 5′-GAAAGGAUCAGUGUCGAUA-3′51, siSEPT2 5′-GCCCUUAGAUGUGGCGUUU-3′70, siSEPT6 5′-CCUGAAGUCUCUGGACCUAGU-3′19, siPIPKIγ 5′-GAGGAUCUGCAGCAGAUUA-3′, siPIPKIγ-i5 5′-CAGAAGGGCUUUGGGUAA-3′36, siPIPKIγ-i3/i5 5′-GGAUGGGAGGUACUGGAUU-3′36. On-Target Plus siRNA smart pools (Dharmacon) were used to silence PIPKIα (L-004780-00-0010), PIPKIβ (L-004058-00-0010), MgcRacGAP (L-008650-00-0005), anillin (L-006838-00-0005), or SEPT7 (L-011607-00-0005). The control siRNA used throughout the study had the following sequence: 5′-GUAACUGUCGGCUCGUGGU-3′.

Generation of antibodies
An antibody specifically recognizing PIPKIγ was raised in rabbits immunized with recombinant His-tagged PIPKIγ (aa 451-668)71. The same recombinant protein was covalently coupled to CNBr-coupled sepharose and used for affinity purification. An antibody specifically detecting SEPT6 was raised in rabbits immunized with a peptide comprising aa 413-327 of SEPT6 (CAGGSQTLKRDKEKKN), and affinity-purified on the same epitope.

Cell lines
HeLa and HEK-293T cells were obtained from American Type Culture Collection (ATCC), and not used beyond passage 30 from original derivation. The genome-edited NRK49F SEPT2-eGFP knock-in cell line has been described previously47. HeLa cells were cultured in Dulbecco`s modified Eagle`s medium (DMEM) containing 1 g/L D-glucose and phenol red (PAN Biotech), supplemented with 10% (vol/vol) heat-inactivated fetal bovine serum (FBS, Gibco), 2 mM L-glutamine (Gibco), 50 μg/mL penicillin-streptomycin (pen-strep, Gibco). Stably transfected HeLa cells were generated by viral transduction, and maintained under selection pressure by additionally supplementing the above-described medium with 1 μg/mL puromycin (Invivogen). HEK-293T cells were cultured in DMEM containing 4,5 g/L D-glucose, phenol red, L-glutamine (Gibco), supplemented with 10% FBS and 50 μg/mL pen-strep. NRK49F SEPT2-eGFP cells were cultured in DMEM containing 4,5 g/L D-glucose (Gibco), supplemented with 10% FBS and 2 mM L-glutamine. All cell lines were cultured at 37 °C and 5% of CO , and regularly tested for mycoplasma contamination.

Generation of an eGFP-SEPT6 knock-in cell line
Endogenous tagging of the SEPT6 N-terminus with eGFP was achieved via the CRISPR/Cas9 technology72. In brief, the primers 5′- CACCGCATCGCTCCTGCGTCCGCCA-3′ and 5′-AAACTGGCGGACGCAGGAGCGATGC-3′ were annealed and subcloned into px458-pSpCas9(BB)-2A-GFP (Addgene) using the BpiI restriction site to generate a guide RNA.

In a second donor vector, the expression cassette was exchanged with the CDS of eGFP inserted between two homology regions (HR) consisting of original genomic sequences ~1000 bp upstream of the SEPT6 ATG (5′ HR) and ~1000 bp downstream of the SEPT6 ATG (3′ HR). The stop codon of eGFP was exchanged with two codons encoding for a Gly-Ser linker. Design and cloning of the donor vector were performed with the NEBuilder assembly tool and NEBuilder HiFi DNA assembly cloning kit, respectively, according to the manufacturer instructions. HeLa cells were co-transfected with px458- pSpCas9(BB)-2A-GFP containing the guide sequence, and with the donor vector. 72 h later, eGFP-expressing HeLa were sorted into 96-well plates at a density of one cell per well, using a fluorescence-activated single cell sorter (BD FACSAria). Growing colonies were expanded and tested for the expression of eGFP-SEPT6 by automated live cell imaging and Western blotting using anti-SEPT6 and anti-GFP antibodies. The expression of eGFP-SEPT6 in selected clones was further validated by siRNA-mediated depletion of SEPT6, by immunoprecipitation of GFP, and by immunocytochemistry.

siRNA-mediated gene silencing
To silence PIPKIα, PIPKIβ, PIPKIγ, and PIPKIγ-i3/i5, two rounds of ~48 h knock-down each were performed with 50 nM siRNA, using JetPRIME (Polyplus) as transfection reagent according to the manufacturer´s instructions. For immunocytochemistry (ICC) and Ultrastructure expansion microscopy (U-ExM, see below), cells were seeded on Matrigel (Corning)-coated glass coverslips in a 12-well plate for the second round of knockdown. For live-cell imaging of eGFP-SEPT6, the second round of knockdown was performed in matrigel-coated 8 well glass-bottom slides (ibidi). Cells were analyzed ~48 h after the second round of transfection. Silencing of SEPT2 and SEPT6 was achieved with one round of knockdown for ~48 h with 100 nM siRNA and JetPRIME as transfection reagent according to the manufacturer´s instructions.

For the co-depletion of PIPKIγ-i3/i5 and OCRL, two rounds of knockdown were performed, with 50 nM siPIPKIγ-i3/i5 + 50 nM siOCRL. In the same experiment, the single depletions of PIPKIγ-i3/i5 and OCRL were performed with 50 nM targeting siRNA + 50 nM siControl, while control cells were treated with 100 nM siControl at each round.

Analysis of the expression of PIPKIγ isoforms
RT-PCRs were performed as described previously73. Briefly, 1 μg of RNA was used with isoform-specific reverse primers for the RT-reaction, and the subsequent PCR was performed with a 32P-labeled forward primer. Products were separated by denaturing PAGE and quantified using a Phosphoimager (Typhoon 9200, GE Healthcare) and ImageQuant TL software. The sequences of the primers used for the initial PCRs and the expected sizes of the amplicons were as follows: Common PIPKIγ forward: GCGCCCGCCACCGACATCTAC; PIPKIγ-i1-3 reverse: CATCTCCCGAGCTCTGGGCCTC (i1=125 nt, i2=210 nt, i3=290 nt); PIPKIγ-i4 reverse: GAGACCAGGACGCGCACAAACCAG (i4=154 nt); PIPKIγ-i5 reverse: CAGACACTGAGCTTCCGGCCGG (v5=195 nt). For isoforms i3 and i5, we further optimized the PCR by using a reverse primer that amplifies a region common to the i3 and i5 isoforms (v3_5rev: AGTCCCCGAGGCGCTC) with the common forward primer (amplicon size 100 nt). To quantify specific knockdown of i3/i5 isoforms, we normalized the i3/i5 product to i1, which was the main isoform.

Plasmid overexpression
Transfection of HeLa was performed with JetPRIME (Polyplus), according to the manufacturer´s instructions. The medium was exchanged after 6 h of transfection, and the cells were processed after 24 h of transfection. For the overexpression of PIPKIγ isoforms, cells were seeded on matrigel-coated glass coverslips in a 12-well plate, and transfected with 0.5 μg of DNA per well.

Transfection of NRK49F SEPT2-eGFP was performed with Lipofectamine 3000 (Thermo-Fisher), following the manufacturer´s instructions. Cells were seeded on glass coverslips in a 6-well plate, transfected on the following day, and used for experiments 48 h post-transfection. For immunoprecipitation assays or for virus production, HEK-293T cells were transfected using calcium phosphate. In brief, DNA was mixed with 0.12 M CaCl in 0,1× TE buffer (1 mM TRIS, 0.1 mM EDTA, pH 8.0) and incubated for 5 min at room temperature. The same volume of 2X HBS (280 mM NaCl, 10 mM KCl, 1.5 mM Na2HPO4, 12 mM dextrose, 50 mM HEPES, pH 7.0) was added while stirring. After 20 min of incubation at room temperature, the DNA solution (1 mL for a 10 cm dish) was added to the cells.

Cytochalasin D treatment and phalloidin staining in NRK49F SEPT2-eGFP
Cells were incubated for 30 min in full medium containing 5 μM cytochalasin D. After a quick rinse with PHEM buffer (60 mM PIPES, 25 mM HEPES, 2 mM MgCl , 10 mM EGTA, pH 6.9), cells were fixed with 4% PFA in PHEM buffer at 37 °C for 15 min. Excess of PFA was quenched with 50 mM NH Cl in PHEM buffer for 10 min on a shaking platform. Cells were washed three times for 5 min on a shaking platform, and stored for imaging, or processed for phalloidin staining.

For phalloidin staining, cells were permeabilized with 0.3% Triton-X100 in PHEM buffer for 5 min. Next, samples were blocked with 4% horse serum and 1% BSA in PHEM buffer for 1 h. Coverslips were incubated with 1 μM AlexaFlour-647-coupled phalloidin (Thermo-Fisher) in 1% BSA/PHEM buffer for 30 min, and then washed three times for 10 min in PHEM buffer on a shaking platform. Coverslips were mounted in Fluoromount G (Invitrogen).

Immunocytochemistry
HeLa cells seeded on matrigel-coated glass coverslips were fixed with 4% PFA/4% sucrose, or with 2% PFA/2% sucrose (for septin stainings) in PBS for 15 min at RT, and subsequently washed three times with PBS. Cells were permeabilized with washing buffer (20 mM Hepes pH 7,2, 150 mM NaCl, 0.1% Triton X-100) for 15 min, and then blocked with goat serum dilution buffer (GSDB: 20 mM Hepes pH 7.2, 150 mM NaCl, 0.1% Triton X-100, 10% goat serum) for 20 min. Incubation with primary antibodies, diluted in GSDB, was carried out at RT for 1 h, and the excess of antibody was removed by three washes of 10 min each with washing buffer. Cells were subsequently incubated with AlexaFluor-conjugated secondary antibodies diluted in GSDB for 1 h at RT. After three washes in washing buffer, coverslips were incubated for 5 min with 1 μg/mL of DAPI in PBS, and mounted on microscope glass slides with Immu-Mount (Thermo-Fisher). For the staining of anillin, goat serum was replaced by donkey serum throughout the protocol.

Plasmalemmal PI(4,5)P was visualized using purified, recombinant pleckstrin homology (PH) domain of PLC-δ1. Cells were fixed with 2% PFA, 2% sucrose + 1% glutaraldehyde (GA) in PBS for 20 min at RT. Excess of fixative was quenched by three washes with 50 mM NH Cl in PBS. Permeabilization and blocking were performed concomitantly with a first round of incubation with 0.25 μg/mL of His -tagged eGFP-PH-PLCδ1 in 0.5% saponin, 1% BSA in PBS for 30 min at RT. Subsequently, cells were incubated (without washing in between) again with 0.25 μg/mL of His -tagged eGFP-PH-PLCδ1 diluted in 1% BSA in PBS for 30 min at RT. Subsequently, coverslips were washed three times with PBS, and incubated with primary antibody (rabbit-anti-GFP, Abcam) in 1% BSA, 10% GS in PBS for 1 h at RT. Excess of antibody was removed by three washes with PBS. The secondary antibody (AlexaFluor 488-coupled goat-anti-rabbit) was diluted in 1% BSA, 10% GS in PBS and incubated at RT for 30 min. Excess of antibody was removed by four washes with PBS, supplemented with 1 μg/mL of DAPI for the final wash before mounting. To visualize PI(4,5)P at the midbody by antibody labeling (mouse-anti-PI(4,5)P, Echelon), fixed cells were permeabilized in 0,5% saponin, 1% BSA in PBS for 30 min at RT. Coverslips were incubated with primary antibodies diluted in 1% BSA, 10% GS in PBS for 2 h at RT. Excess of antibodies was removed by three washes in PBS, and coverslips were incubated with AlexaFluor-coupled secondary antibodies diluted in 1% BSA, 10% GS in PBS for 1 h at RT. Coverslips were subsequently washed three times with PBS, once with PBS supplemented with 1 μg/mL of DAPI, and finally mounted.

Cells synchronization
For microscopy-based assays, 2 mM thymidine was applied in full medium to stall the cells at S-phase. 24 h later, thymidine was removed by four washes of 1 min with PBS, and cells were incubated for 7,5 h in fresh medium. Then, fresh medium supplemented with 20 ng/mL of nocodazole was added. After 4 h, cells were carefully washed four times for 1 min with full medium pre-warmed at 37 °C, and subsequently allowed to proceed to cytokinesis by incubation for 1.5 h in full medium, before fixation. For live cell imaging, the nocodazole block was omitted.

For immunoprecipitation, the nocodazole block was applied overnight by supplementing the medium with 40 ng/mL of nocodazole. The following day, cells were collected by mitotic shake-off and washed four times in full medium by centrifugation at 300×g for 5 min. Cells were then re-plated in full medium and allowed to proceed to cytokinesis for 90 min. Cells were collected by gentle shake-off and harvested by centrifugation at 300×g for 5 min. Before lysis, cells were washed once in PBS. Cells stalled at the S-phase for 48 h served as controls and were harvested by trypsinization.

Microscopy and image analysis
Immunostained HeLa cells were routinely imaged with a Zeiss confocal spinning disk microscope (Yokogawa CSU22, Hamamatsu EMCCD camera), using a 60× immersion oil objective (1.4 NA). At least 12 images were acquired per condition, per experiment. For each image of cytokinetic cells, a stack of 21 pictures within the z-plane (z-stack), with a spacing of 0.2 μm was acquired. Cells stained with purified His -tagged eGFP-PH-PLCδ1 domain (Supplementary Fig. 1e), antibodies recognizing PI(4,5)P (Fig. 3d), and knock-in eGFP-SEPT6 cells (Supplementary Fig. 4c) were imaged within a single z-plane by focusing on the plasma membrane, or on prominent septin filaments, respectively. For the experiment depicted in Fig. 1 and Supplementary Fig. 1b, semi-automated epi-fluorescent imaging was conducted with a Nikon Eclipse Ti microscope (illumination: CoolLED, pE4000, prime95B sCMOS camera) operated by NIS-Elements software, using a 20× air objective (0.75 NA). Tilescans of 1.3 mm2 were produced by stitching together images automatically acquired around a chosen point. Four tilescans were acquired per condition, per experiment. Epi-fluorescent pictures of HeLa cells transfected with PIPKIγ isoforms (Supplementary Fig. 2c) were acquired with the same microscope, using a 40× immersion oil objective (1.3 NA). Live cell imaging of eGFP-SEPT6 cells was performed on a spinning disk Nikon Eclipse Ti microscope (Yokogawa CSU-X1 and EMCCD Camera), operated by NIS-Elements software, with a 40x air objective (0.95 NA). Imaging was initiated 7, 5 h after thymidine release, and was carried out overnight, with a frame rate of 10 min. Cells were kept in full medium at 37 °C and 5% CO , and pictures were acquired within a single z-plane that was set at the beginning by focusing on septin fibers of pre-mitotic cells, and kept by an autofocus system. NRK49F SEPT2-eGFP cells and gels for U-ExM were imaged with an Olympus spinning disk microscope (Yokogawa CSU-X1, Hamamatsu C11440 camera), using a 60x immersion oil objective (1.42 NA). Cells depicted in Fig. 2d, e were imaged within a single z-plane by focusing on septin fibers. For cells depicted in Fig. 2f, a z-stack with a spacing of 0.3 μm was acquired. For U-ExM, 20 images were acquired per condition, per experiment. For each image, a z-stack of 21 layers with a spacing of 1 μm was acquired. For imaging of SiR-tubulin, cells were seeded into Matrigel-coated Ibidi 8-well glass-bottom dishes and incubated with 100 nM SiR-tubulin (Spirochrome) in full media overnight. Imaging was performed in a live-cell incubator with 5% CO2 (OkoLab) heated at 37 °C, on a Nikon TiE2 (Nikon) equipped with a confocal spinning disk unit (CSU-W1, Yokogawa) with a PLAPO λ 40x/0.95 NA air objective without additional magnification. The microscope was equipped with two sCMOS cameras (pco.edge, 4.2 bi, 6.5 μm/pixel, 2048×2048 pixel) and controlled with NIS-Elements software (Nikon). Images were taken with the following sequential fluorophore settings: DAPI (Ex.: 405 nm; Em.: 420–460 nm), EGFP/AF488 (Ex.: 488 nm; Em.: 500–550 nm), JFX554 (Ex.: 561 nm; Em.: 574–626), AF647 (Ex.: 638 nm; Em.: 670–746-nm). Live imaging was performed in the AF647 channel with 10 min frame interval, and videos were analyzed by measuring the time from initiation of mitosis (marked by cell rounding) until completion of furrow ingression (marked by the appearance of a thin microtubule bridge), and thereafter until microtubule cut (marked by a sudden retraction of the microtubule bundle from the ICB into one of the daughter cells).

Image processing and analysis were performed with the open-source software Fiji (ImageJ).

Quantifications depicted in Fig. 1b–e were performed by identifying and manually counting mitotic cells displaying a mitotic spindle, or a cytokinetic bridge, as schematized in Fig. 1a. This number was then divided by the total number of nuclei, identified via a macro and serving as the total number of cells in the picture.

The qualitative assessment of anillin at the ICB was conducted on maximum intensity projections of z-stacks. For measuring the intensity of the acetylated tubulin bridge, average intensity projections of the z-stacks were generated, and the acetylated tubulin channel was segmented in order to obtain regions of interest (ROI) outlining the acetylated tubulin bridges. Subsequently, the fluorescence intensity of acetylated tubulin was measured as integrated density within the obtained ROI on average intensity z-projections, after background subtraction. The intensity of PRC1 bridges, MKLP1, and MgcRacGAP dots was measured the same way, in regular confocal or expanded samples. The length of the acetylated tubulin bridges was measured as the max Feret diameter of the ROI outlining the acetylated tubulin bridges. Septin enrichment at the acetylated tubulin bridge was determined by dividing the intensity of SEPT2 in a ROI outlining the acetylated tubulin bridge by the intensity of SEPT2 in a ROI outlining the whole dividing cell (drawn by hand); here, as well, measurements were carried out on average intensity z-projections, after background subtraction. In quantifications depicted in Supplementary Fig. 1f, the intensity of PI(4,5)P per cell area was measured as the mean gray value of the eGFP-PH-PLCδ1 fluorescence in the ROI outlining single cells (drawn by hand), after background subtraction. Intensity line scan analysis, as depicted in Fig. 2g, was performed on maximum intensity projections of the z-stacks. Midbody PI(4,5)P and CIT-K levels, as depicted in Fig. 3e, f were determined by placing an equally sized ROI of 3 μm in diameter on the CIT-K signal at the midbody. Intensities were measured upon background subtraction.

For all displayed images, brightness and contrast were adjusted equally for different conditions, unless otherwise indicated.

Ultrastructure expansion microscopy (U-ExM)
Samples were processed using the U-ExM method53. Abbreviations used: FA (Formaldehyde), AA (Acrylamide), BIS (N,N′-Methylenebisacrylamide), TEMED (N,N,N′,N′-Tetramethylethylenediamine), APS (Ammonium persulfate), SA (Sodium acrylate). HeLa cells (seeded on 18 mm matrigel-coated coverslips) were rinsed with PHEM buffer (60 mM PIPES, 25 mM HEPES, 2 mM MgCl , 10 mM EGTA, pH 6.9), then fixed in 4% PFA, 0.1% Triton X-100 in PHEM at 37 °C for 15 min. Fixed coverslips were rinsed in PHEM buffer again and transferred into 1 mL of anchoring solution (0.7% FA, 1% AA in PBS). Samples were incubated for 16 h at RT. U-ExM monomer solution (19% (wt/wt) SA, 10% (wt/wt) AA, 0.1% (wt/wt) BIS in PBS) was prepared 1 day before the gelation and stored at −20 °C until use. 90 μL of monomer solution was mixed with 5 μL of 10% TEMED and 5 μL of 10% APS (0.5% APS and TEMED in the final monomer solution) and placed on Parafilm in a drop in a pre-cooled humid chamber. The coverslips were placed on the drops facing downwards and incubated on ice for 5 min. For gelation, coverslips were incubated at 37 °C for 1 h in the humid chamber. Gels (still on coverslips) were placed in 2 mL denaturing buffer (200 mM SDS, 200 mM NaCl, 50 mM Tris, pH 9) in a six-well plate for 15 min at RT with agitation. For denaturation, gels were transferred into 15 ml centrifuge tubes filled with 3 mL of denaturation buffer and incubated at 95 °C for 1 h. Subsequently, the gels were washed several times with MilliQ water and then expanded in 10 cm dishes in MilliQ water. The water was exchanged twice after 30 min. Finally, gels were left to expand in water overnight at 4 °C.

Gels were measured with a caliper the following day to calculate the expansion factor. Gels were cut into 2×2 cm squares, and the squares were incubated twice in PBS for 15 min. The shrunken gels were stained with antibodies diluted in 2% BSA/PBS. To this end, each gel piece was placed in a 12-well plate, covered with 500 μL of antibody solution, and incubated for 3 h at 37 °C and at 120 rpm. Gels were then washed three times for 20 min with PBS-T (PBS supplemented with 0.1% Tween 20) in a 6-well plate with gentle agitation. Secondary antibody incubation was executed as indicated above for 2.5 h. Gels were then washed three times for 20 min with PBS-T in a 6-well plate under gentle agitation. Final expansion was done in MilliQ water in 10 cm dishes, exchanging the water at least two times after 30 min, and letting the expansion plateau by an overnight incubation at 4 °C. For imaging, gels were mounted in imaging dishes (Zell-Kontakt) with 2% agarose gels. To calculate the scale, the pixel size of the camera was divided by the measured gel expansion factor (~4,2).

Cell lysates
Cells were trypsinized, washed with PBS, and lysed for 15 min on ice in lysis buffer (20 mM HEPES, 100 mM KCl, 2 mM MgCl , 1% Triton X-100, pH 7,4) supplemented with protease inhibitor cocktail (Sigma-Aldrich) and 1 mM PMSF. Lysates were cleared by centrifugation at 17,000 g for 15 min at 4 °C, and the protein concentration was determined with Bradford reagent (Sigma-Aldrich). Lysates were subsequently denatured in Laemmli sample buffer for 5 min at 95 °C. For each sample, 10–30 μg of protein were resolved by SDS-PAGE and analyzed by western blot (see below).

Immunoprecipitation assay
HEK-293T cells seeded in 10 cm petri dishes were transfected at 70% confluency with 5 μg of plasmid encoding mCherry-PIPKIγ or mCherry alone and 5 μg of myc-tagged SEPT7 or SEPT9, using the calcium phosphate method. Twenty-four hours later, cells were trypsinized, washed once with PBS, and lysed at 4 °C for 15 min in lysis buffer (20 mM HEPES, 100 mM KCl, 2 mM MgCl , 1% Triton X-100, pH 7,4) supplemented with protease inhibitor cocktail (Sigma-Aldrich), 1 mM PMSF, and phosphatase inhibitors cocktails 2 and 3 (Sigma-Aldrich). Lysates were cleared by centrifugation at 17,000×g for 15 min. 0.6 mL of the resulting supernatant (containing 2–4 mg of proteins) was supplemented with 15 μL of RFP-Trap magnetic particles (ChromoTek), and incubated for 2.5 h at 4 °C on a rotating wheel. Beads were washed three times with lysis buffer and a fourth time with lysis buffer without detergent. Proteins bound to the beads were eluted by boiling in 60 μL of Laemmli sample buffer for 5 min, resolved by SDS-PAGE, and analyzed via immunoblot.

For immunoprecipitation experiments cell lysates derived from HeLa cells arrested in S-phase by thymidine treatment (obtained upon trypsinization), or from HeLa cells in cytokinesis (obtained by gentle pipetting) were incubated with 5 μg of rabbit-anti-human control, of rabbit-anti-SEPT2, or of rabbit-anti-PIPKIγ antibodies, bound to protein A/G magnetic beads (PierceTM) at 4 °C for 3–4 h on a rotating wheel. Beads were washed four times in lysis buffer (20 mM HEPES, 100 mM KCl, 2 mM MgCl , 1% Triton X-100, pH 7,4) and once in lysis buffer devoid of detergent. Retained proteins were released by boiling in 90 μL of Laemmli sample buffer for 5 min, resolved by SDS-PAGE, and analyzed by Western blotting.

GST pulldown assay
Expression and purification of GST-tagged PIPKIγ tails (or of GST alone, used as control) was performed essentially as described previously71. All purification steps were carried out on ice. Briefly, the pellet of 250 mL expression culture was resuspended in 35 mL of PBS, sonicated, supplemented with 150 U of benzonase (Sigma-Aldrich), incubated for 10 min, supplemented with 1% Triton-X-100, sonicated, and incubated again for 10 min. The bacterial lysate was cleared by centrifugation at 50,000×g for 15 min. The supernatant was recovered, incubated with 500 μL of GST-Bind ResinTM (EMD Millipore), and incubated for 2 h by end-over-end rotation. The resin was washed three times with 20 mL of PBS, and finally resuspended in 700 μL of PBS. Quantity and integrity of the purified fusion protein were determined by SDS-PAGE, followed by Coomassie Blue.

For pulldown experiments, mouse brain extracts were obtained by homogenizing four brains in homogenization buffer (20 mM HEPES, 320 mM sucrose, pH 7.5) containing complete EDTA-free protease inhibitor cocktail (Roche). The homogenate was centrifuged at 1000×g for 15 min at 4 °C. The supernatant was recovered, supplemented with 1% Triton X-100, 100 mM KCl, 2 mM MgCl2, and incubated on ice for 10 min. The lysate was cleared by centrifugation at 17,000×g for 15 min, and subsequently at 178,000×g for 15 min (4 °C). The supernatant was recovered and used at a concentration of 14 mg protein/mL. Pulldown experiments were performed by incubating 1 mL of protein extract with 70 μg of GST-fusion protein or of GST for 3 h at 4 °C by end-over-end rotation. The samples were subsequently washed four times with buffer containing 20 mM HEPES, 100 mM KCl, 2 mM MgCl2, 1% Triton X-100, pH 7.4, and once in the same buffer without detergent. Proteins were eluted from the beads by boiling for 5 min in 100 μL of Laemmli buffer, and analyzed by Western blotting. Chemiluminescent signals were detected using the ChemieDoc MP Imaging System (BioRad) controlled by the Image Lab 6.0.1 software. Fluorescent signals were detected using the LI-COR Odyssey Fc imaging system controlled by the Image Studio software.

Statistics and reproducibility
All quantitative data were derived from at least three independent experiments and are presented as means±standard deviation (SD). GraphPad Prism version 9.2 software was used for statistical analysis. Unpaired two-tailed t-test was applied to compare two groups. One-way ANOVA followed by Dunnett´s or Tukey´s multiple comparison test was used to compare more than one experimental group to a control group. When the control group was set to 1 by normalization, one sample two-tailed t-test was applied for comparing one or more experimental groups to the control. The level of significance is indicated in the figures by asterisks (*P<0.05; **P<0.01; ***P<0.001; ****P<0.0001), and detailed in the Source Data files as exact P value.

Affinity purification experiments, as shown in Fig. 2b were performed three independent times with PIPKIγ-i2, -i3, -i5, twice including also PIPKIγ-i5 ΔSB, and yielded similar results. Immunoprecipitation experiments, as illustrated in Figs. 2c and 5h, i, were repeated several times with similar results, but not in all experiments all potential interaction partners were analyzed, and not in all experiments lysates of HeLa cells in S-phase were included. In summary, in immunoprecipitates of PIPKIγ, septins were detected in five independent experiments, CIT-K in seven experiments, and centralspindlin in five experiments. In immunoprecipitates of SEPT2, centralspindlin was detected in six independent experiments, anillin in seven experiments, PRC1 in nine experiments, and PIPKIγ in two experiments. Two independent experiments were performed to analyze the co-localization of SEPT2-eGFP with F-actin in genome-edited NRK49F cells, and yielded similar observations to those depicted in Fig. 2d. Co-localization of PIPKIγ-variants with SEPT2-eGFP as was assessed in four independent experiments, with a similar outcome as depicted in Fig. 2e. Results as shown in Fig. 2f were reproduced in three independent experiments. The localization of mCherry-PIPKIγ-i5 was correlated with SEPT2 and anillin in three independent experiments, with similar results as depicted in Fig. 3a. Please see Source Data for additional information on the reproducibility of results shown in Supplementary Figs.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "Russo et al. present how PIPKlɣ, an enzyme synthetizing PI(4,5)P2, interacts with partners at the intercellular bridge and\nmidbody during cytokinesis. The results demonstrate that the isoform PIPKIɣ has a key role at the midbody as compared\nwith other isoforms α and β. The interplay between key proteins (septin, anillin, centralspindlin) at the midbody and PIPKIɣ is\ndescribed to propose a model where the synthesis of PI(4,5)P2 tunes the organization of the midbody throughout\ncytokinesis. Russo et al. have done some interesting work to understand the mechanisms occurring during cytokinesis.\nHowever, some control experiments would be welcome to make their points more convincing. Hence, I would recommend\nsome revisions before the manuscript can be accepted in Nature Communications.\nMajor revisions\n-In most experiments, PIPKIɣ is depleted, while the expression, localization of possible partners are assayed. Conversely,\ndepleting septins, anillin, or centralspindlin and describing the expression level and localization of PIPKIɣ would be key to\nactually understand how PIPKIɣ is controlled and dependent on those key partners. Besides, the authors state, at least\ntwice, their assays demonstrate a “septin-dependent recruitment of PIPKIɣ” while they actually describe a PIPKIɣ-\ndependent recruitment of septins.\n-Testing the role of PIPKl on myosin II recruitment would be valuable as well given myosin is involved in cytokinesis.\n-To demonstrate synergistic roles of the different PIPKl, particularly on the distribution of PI(4,5)P2, depletions of\ncombinations of the isoforms would be worthwhile. Have they authors attempted to deplete combinations of 2 or all 3\nsimultaneously?\n-On Figure 1.h, upon depletion of PIPKlɣ, septins can still be visualized bound to the plasma membrane as well as within\nthe midbody. Was this a frequent observation? Can the authors comment on this? Besides, depletion of PIPKlβ seems to\nalso disrupt septin localization given that septins can be seem as “clusters”or point-like densities. Can the authors comment on\nthis?\n-While using expansion microscopy septins seem to localize and organize as rings on both sides of the midbody, this\nlocalization is not seen using conventional microscopy. Instead septins seem to colocalize with microtubules in a bundle-\nlike organization. Why would that be different?\n-From the literature, it is considered that anillin would recruit septins. However, in this study, the authors conclude that\nseptins would promote anillin recruitment through PIPKI at the midbody. Can the authors comment on this observation?\n-Figure 4: Depletion of PIPKI induce mis-localization of septins and disruption, to some extent, of the microtubule spindle.\nIs it a direct effect or does it occur through a PRC1 dependent effect? Can the authors possibly test or at least comment on any\ndirect interaction between PRC1 and septins, for instance? Besides, did the authors consider testing how microtubule\ndisruption would affect septin and PIPKI distribution in the cell?\nMinor revisions\n-In different figures, the displayed images are too small (see, for instance, Figures 1.f, 3.d, 3.g, Supl. 1.b, Supl. 4.d, 4.e…).\nEnlargements would thus be welcome for the evidences to be more convincing.\n-It would be worthwhile to show images (in supplementary) corresponding to the phenotypes described in Figure 1.d and 1.e\n(multipolar spindles and bridges, respectively).\n-Can the authors comment on the effect of Thymidine, used for cell synchronization? Can it affect the ultrastructure, stability\nof the midbody and/or the recruitment of specific proteins?\n-P7, l196: The authors should specify that the affinity purification was performed from a mouse brain lysate.\n-Figure 2e.: Some of the cells are not labelled with m-Cherry-PIPKlɣ while a septin signal is visible. Can the authors give an\nexplanation?\n-Figure 2.f: Did the authors perform the same experiment testing for the i3 isoform? Did they obtain the same phenotype as\nfor i5 mutant?\n-Supplementary Fig.3: The numbering for the different panels differs from the font/numbering used for all other figures.\nThis should be homogenized.\n-Page 9, top (l 246-253): The described result is not displayed or it is unclear ?\n-Figure 3.d: On the siOCRL panel, the signal from citron kinase seems much higher than in the panels above and below.\nHowever the quantification (3.f) indicates a rather constant intensity depending on the conditions. Can the authors comment\nor correct accordingly?\n-Supplementary fig. 4.g: The septin pattern observed for i5 WT looks different from others and septins seem to localize in the\nmidbody as well. Can the authors comment on this?\n-Figure 5.k: The signals for MKLP1 and septins from an IP via PIPKIɣ are rather weak. Can the authors comment on this?\nTesting for centralspindlin, have the authors also assayed RacGap1?", "reviewer_2": "The manuscript submitted by Russo and colleagues addresses the question of how PIP2 is generated at the intercellular\nbridge during mammalian cytokinesis and what effect it has on cytokinetic progression. They report that coupling of the PI-5-\nP kinase PIPKIγ with septins is required to stabilize the centralspindlin complex at the midbody/Flemming body in late\ntelophase. This study addresses an important and unresolved question in the field and is thus of significant interest to cell\ndivision researchers. Additionally, it reveals an interesting coupling of the septin cytoskeleton to lipid synthesis. However, a\nnumber of clarifications are required.\nMajor points:\n1. A major deficiency is the manuscript are a lack of rescues for siRNA-mediated depletions in multinucleate experiments\ndescribed in Figures 1 and Supp Figure 3D. Given that the phenotypes are small in absolute terms, these rescues are\nparticularly important to establish that the relatively small increases observed for cytokinesis failure are not due to off-target\neffects. While the rescues shown in Fig 3i are appreciated, they are not direct evidence of a rescue of cytokinesis failure.\nRescues would preferably be undertaken with strategies that avoid over-expression.\n2. The phenotype induced by PIPKIγ disruption (either by siRNA alone or with expression of mutants) requires more\ndefining. If PIPKI  is important for telophase progression, is there a measurable delay in telophase? Does abscission take\nlonger? Some timing experiments could help clarify\n3. Some terms used in the manuscript are unclear and ambiguous. The common one of ‘compact’ to describe anillin\nlocalization is unclear and does not reflect what can be seen in the images. (Fig 1,3). Nor does this reflect previous studies\nthat defined anillin dynamics in the intercellular bridge (El Amine et al. 2013, Renshaw et al. 2014). These two studies\ncharacterized in detail anillin dynamics and define common organizational states that reflect different phases of intercellular\nbridge assembly. Assessing if and/or when these previously defined patterns of anillin localization are disrupted by the\nvarious perturbations performed in this study will make comparisons across studies much easier.\n4. Related to point 3, in many cases the micrographs being compared appear to be at different stages of intercellular bridge\ndevelopment (i.e. that the authors are comparing two ICBs of differing ages). This occurs frequently in Figs 3 , S3 and 4.\nIncluding tubulin staining to the experiments would help clarify these issues as the degree of microtubule compaction has\nbeen used to assess the intercellular bridge age thereby allowing more direct “apples to apples” comparisons. This is a very\nimportant point with respect to the conclusions made and should be rigorously addressed.\nMinor points:\nA. In the introduction the Piekny and Maddox review is extensively cited. While this is a good review it is 15 years old and\nmuch has been learnt subsequently about anillin, who it binds to and how it functions. It would be useful to newcomers to the\nfield if more up to date references about specific points were used.\nB. The biology of septins needs a more thorough treatment, again with up to date references. While it is said that septins\nhetero-oligomerize, the hexamer/octamer distinction is bound to be confusing to non-experts and should be better\ndeveloped. In addition, in the results section (line 170) the authors state that septins bind to the C-term of anillin. While this is\nthe conclusion drawn from this 2000 paper, subsequent work suggests a more complex relationship. This combined with a\nbetter discussion of hexamers and octamers would bring the paper up to date and allow a subsequent more detailed\ndiscussion (and perhaps experimental plan) of the underlying mechanism at play.\nC. In the results, a simple statement that cells were synchronized by double thy + nocodazole treatment should be added to\nthe main text to aid the reader.\nD. Experiments demonstrating the localization of anillin and septins in PIPKIγ-depleted cells across the stages of\ncytokinesis (as shown for control cells in Fig 3a) would be helpful. As the authors state, anillin and septin recruitment to the\nequatorial membrane prior to furrow ingression are known to be dependent on PI(4,5)P2. However, the manuscript only\nshows disrupted telophase localization of anillin in knockdown/mutant conditions. This may result from deficiencies in\nrecruitment to the furrow/ICB in anaphase-early telophase as opposed to the action of PIPKIγ later in telophase.\nE. What effect does knockdown of PIPKIα & β have on PI(4,5)P2 levels in anaphase and telophase cells? The results of Fig\n3 would suggest that PIPKIγ is responsible for the majority of PI(4,5)P2 present in cytokinetic cells. Is the remaining\nPI(4,5)P2 is sufficient to promote initial anillin/septin ICB recruitment?\nF. Fig 4: Measurements comparing the width of ICB bridges across conditions should be performed. If as the authors argue,\ndepletion of PIPIKIγi3-5 perturbs the development of the ICB such that its microtubules do not compact as much as control,\nthen the width of acetylated tubulin staining will better bolster this point than measurements of ICB length.\nG. Fig5hi: I do not think it’s appropriate to measure fluorescence intensities across different expanded samples unless steps\nwere taken to ensure some normalization of expansion factors and a comparison to some constant. The obvious differences\nin intensity presented in panel g are more than sufficient to support the authors’ claims.\nH. In S3G: the i5ΔsupSB panel contains a cell that appears to be at an earlier stage of cytokinesis compared to the other\ncells in this panel (based on less compaction of tubulin staining). It further also looks to be multinucleated. Is this the norm?\nIf yes this would have implications in interpretation.\nI line 76 PI(4,5)2 should be PI(4,5)P2", "reviewer_3": "This manuscript studies the spatiotemporal control of PI(4,5)P2 during cytokinesis and reports a key role for two splice\nisoforms of PIPKIgamma. The authors demonstrate that PIPKIg is required for cytokinesis progression and for the\norganization of anillin and septins at the intracellular bridge (Figure 1). They furthermore identify two splice isoforms (i3 and\ni5) of PIPKIg that can interact with septins (Figure 2)and demonstrate that these are cricital for proper anillin accumulation\nand PI(4,5)P2 synthesis near the midbody (Figure 3), as well as for the recruitment of septin to the intracellular bridge\n(Figure 4) and the association of the centralspindlin complex tot the midbody (Figure 5). Based on these results, the authors\npropose a model in which septins recruit PIPKg to the cleavage furrow and generates local PI(4,5)P2, whereas later on\nseptin-bound PIPKg create even more local PI(4,5)P2 that contributes to septin retention and subsequent microtubule\nstabilization. Overall, the data is convincing and the writing is clear. Nonetheless, I do have a number of comments that need\nto be properly addressed to make the manuscript ready for publication.\n1. I found the authors conclusions on the interdependence of septins and PIPKIg unclear. Initially, the authors postulate that\nseptin recruits PIPKIg to the midzone, whereas later on they suggest that PIPKIg is key for proper localization of SEPT6,\nincluding its localization to microtubules. The text and cartoons should be improved to clarify how the authors think about\nthis interdependence.\n2. In addition, the whole interplay between septins, PIPKIg and microtubule stabilization is not well developed and therefore\nnot very convincing. How does production of PI(4,5)P2 helps to enrich septins on microtubules?\n3. The title and discussion put a lot of emphasis on nanoscale synthesis, but it is unclear if this means anything else than\nlocalized, restricted synthesis. Of course, if an individual protein modifies lipids this can always be considered nanoscale\nsynthesis, but the current formulations are raising the expectations that the authors have discovered nanosized membrane\nregions that carry specific modification. This is not at all reflected in the presented data.\nMinor comments\n4. Figure S3 – panel labels are inconsistent with all other figures\n5. Figure 2f – The merge images of SEPT2 and PIPKg are confusing and would be more clear if presented as separate\nimages. The green is not clear in the WT situation."}, "author_response": "Detailed response to the referees\nWe would like to thank the editor(s) as well as all referees for their careful reading of our\nmanuscript and for their thoughtful suggestions. We were very happy to learn that all three\nreviewers and the editor agree on the high general interest and timeliness of our study and we\nwould like to thank them for their strong support to publish our findings in Nature\nCommunications. We have now conducted a number of new experiments and made textual\nchanges to our manuscript that fully address all questions and concerns raised in the initial\nround of review. We have also tackled all minor points raised as detailed in our response below\n(in blue).\nReviewer #1\nRusso et al. present how PIPKlɣ, an enzyme synthetizing PI(4,5)P2, interacts with partners at\nthe intercellular bridge and midbody during cytokinesis. The results demonstrate that the\nisoform PIPKIɣ has a key role at the midbody as compared with other isoforms α and β. The\ninterplay between key proteins (septin, anillin, centralspindlin) at the midbody and PIPKIɣ is\ndescribed to propose a model where the synthesis of PI(4,5)P2 tunes the organization of the\nmidbody throughout cytokinesis. Russo et al. have done some interesting work to understand\nthe mechanisms occurring during cytokinesis. However, some control experiments would be\nwelcome to make their points more convincing. Hence, I would recommend some revisions\nbefore the manuscript can be accepted in Nature Communications.\nMajor revisions\n- In most experiments, PIPKIɣ is depleted, while the expression, localization of possible\npartners are assayed. Conversely, depleting septins, anillin, or centralspindlin and describing\nthe expression level and localization of PIPKIɣ would be key to actually understand how PIPKIɣ\nis controlled and dependent on those key partners. Besides, the authors state, at least twice,\ntheir assays demonstrate a “septin-dependent recruitment of PIPKIɣ” while they actually\ndescribe a PIPKIɣ-dependent recruitment of septins.\nWe thank the reviewer for this valuable suggestion.\nWe have analyzed expression levels of PIPKI upon depletion of key regulatory proteins\ndescribed in our study. Our novel Supplementary Figure 5d demonstrates that global PIPKI\nlevels are decreased upon depletion of anillin or MgcRacGAP. This indicates that anillin- and\nMgcRacGAP-mediated midbody tethering to the plasma membrane is important for PIPKI\nstability (Kechad et. al., 2012, PMID 22226749; Lekomtsev et al., 2012, PMID 23235882). We\nalso assessed the localization of mCherry-tagged PIPKI-i5 (WT) upon depletion of\nMgcRacGAP, or of anillin. As shown in our novel Supplementary figure 5e, under both\nknockdown conditions kinase recruitment to the ICB is significantly impaired, again in line with\ncentralspindlin´s and anillin´s role in midbody tethering.\nBy contrast, depletion of SEPT7, a septin paralogue required for septin filament stability, does\nnot significantly alter global expression levels of PIPKI (novel Supplementary Figure 5d).\nNonetheless, upon loss of SEPT7 mCherry-PIPKI-i5 (WT) is no longer recruited to the ICB\nduring cytokinesis (novel Supplementary figure 5e). This is in support of our conclusion that\nkinase recruitment depends on the spatiotemporally controlled deposition of septins at the ICB.\nYet, we agree that at the current stage it is difficult to unequivocally claim “septin-dependent\nrecruitment of PIPKI\" and more carefully rephrased throughout the manuscript (“coincident\nrecruitment of PIPKI and septins”).\n- Testing the role of PIPKl on myosin II recruitment would be valuable as well given myosin is\ninvolved in cytokinesis.\nAs displayed in our novel Supplementary Figure 3h phospho-myosin-light chain recruitment\nto the ICB is unimpaired.\n-To demonstrate synergistic roles of the different PIPKl, particularly on the distribution of\nPI(4,5)P2, depletions of combinations of the isoforms would be worthwhile. Have they authors\nattempted to deplete combinations of 2 or all 3 simultaneously?\nWe do agree that PIPKI and of PIPKI might play synergistic roles during furrow ingression.\nBased on the observation that exclusively depletion of PIPKI (and not depletion of PIPKI)\nresulted in late mitotic defects during cytokinesis - the main focus of our study - we did not\nconsider to deplete PIPKI and I in parallel. Please also see our novel data presented in\nSupplementary Figure 3g, which demonstrate that depletion of PIPKI-i3/i5 does not\nsignificantly impair furrow ingression, but rather inhibits timing of the microtubule cut.\n- On Figure 1.h, upon depletion of PIPKIɣ, septins can still be visualized bound to the plasma\nmembrane as well as within the midbody. Was this a frequent observation? Can the authors\ncomment on this? Besides, depletion of PIPKlβ seems to also disrupt septin localization given\nthat septins can be seem as “clusters” or point-like densities. Can the authors comment on\nthis?\nUpon depletion of PIPKI septins (and anillin) randomly distribute at the plasma membrane, in\nmost cases not at the ICB itself, but rather at sites adjacent to it. Of note, the ICB itself suffers\nfrom multiple defects (reduced length: Figure 4d, reduced stability: Figure 4c, impaired MT\nbundling: novel Figure 4e). Septin clusters may also occur at plasmalemmal sites underneath\nthe ICB, as the daughter cells are not so well separated.\nIndeed, we also observe a similar tendency of septin dispersal upon depletion of PIPKI as\ndepicted in Figure 1i, probably due to the known function of PIPKI in furrow ingression\n(Emoto et al., 2005, PMID 16162509). However, this effect was not significant.\n- While using expansion microscopy septins seem to localize and organize as rings on both\nsides of the midbody, this localization is not seen using conventional microscopy. Instead\nseptins seem to colocalize with microtubules in a bundle-like organization. Why would that be\ndifferent?\nAt late stages of cytokinesis we find septins prominently enriched along the ICB, closely\naligning with microtubule bundles. We suppose that the resolution limit of conventional\nmicroscopy does not allow to distinguish this strong staining from overlapping septin rings. Yet,\nwe would like to indicate that at earlier stages, when septins start to translocate onto\nmicrotubules, we find septins also organized in ring-like structures at the midbody (see for\ninstance Figure 3a, Supplementary Figures 3a/4g).\n- From the literature, it is considered that anillin would recruit septins. However, in this study,\nthe authors conclude that septins would promote anillin recruitment through PIPKI at the\nmidbody. Can the authors comment on this observation?\nIt is generally considered that anillin recruits septins to the cleavage furrow, and remains\nassociated with septins during cleavage furrow ingression. Based on super-resolution imaging\nRenshaw et al. (2014, PMID 24451548) reported that at later stages anillin associates with the\nmidbody, and from there follows septins into ring structures flanking the midbody. They also\nreported that this translocation is dependent on its capability to associate with septins.\nBased on our observations we conclude that depletion of PIPKI-i3/i5 reduces the availability\nof PI(4,5)P at the midbody (Figure 4d/e), and prevents stable retention of anillin. Instead,\n2\nanillin follows septins to aberrant PI(4,5)P -enriched sites in the periphery of the ICB.\n2\n- Figure 4: Depletion of PIPKI induce mis-localization of septins and disruption, to some extent,\nof the microtubule spindle. Is it a direct effect or does it occur through a PRC1 dependent\neffect? Can the authors possibly test or at least comment on any direct interaction between\nPRC1 and septins, for instance? Besides, did the authors consider testing how microtubule\ndisruption would affect septin and PIPKI distribution in the cell?\nWe thank the reviewer for this valuable comment.\nIn updated Figure 5j we demonstrate that PRC1 co-purifies with SEPT2 in\nimmunoprecipitation experiments. Further, in our novel Supplementary figure S5c we show\nthat overexpression of eGFP-tagged PRC1 induces strong microtubule bundling in interphase\ncells, and that these microtubule bundles extensively recruit septins. We conclude that septins,\nPRC1 and centralspindlin act in concert to promote microtubule bundling along the ICB.\nConsistently, depletion of PIPKI-i3/i5 and the concomitant loss of septins from the ICB triggers\ndefects in microtubule bundling, as quantified in our novel Figure 4e.\nMinor revisions\n- In different figures, the displayed images are too small (see, for instance, Figures 1.f, 3.d,\n3.g, Supl. 1.b, Supl. 4.d, 4.e…). Enlargements would thus be welcome for the evidences to be\nmore convincing.\nWe apologize for having displayed several images too small, and enlarged them.\n- It would be worthwhile to show images (in supplementary) corresponding to the phenotypes\ndescribed in Figure 1.d and 1.e (multipolar spindles and bridges, respectively).\nOur novel Supplementary Figure S2d shows blowups of multipolar spindles and bridges.\n- Can the authors comment on the effect of Thymidine, used for cell synchronization? Can it\naffect the ultrastructure, stability of the midbody and/or the recruitment of specific proteins?\nThymidine interferes with DNA synthesis, and its application arrests cells in early S-phase. Of\nnote, thymidine was thoroughly washed out to release cells from this block. A second block\nwas then imposed by adding nocodazole, to arrest cells in metaphase. Again, nocodazole was\nthoroughly washed out to let cells progress to cytokinesis. We expect that no excess of\nthymidine or nocodazole was present any more at the time point of fixation.\n- P7, l196: The authors should specify that the affinity purification was performed from a mouse\nbrain lysate.\nWe adapted the Results section accordingly.\n- Figure 2e.: Some of the cells are not labelled with m-Cherry-PIPKlɣ while a septin signal is\nvisible. Can the authors give an explanation?\nCells depicted in Fig. 2e are genome-edited to express SEPT2-eGFP under its endogenous\npromoter. mCherry-tagged kinase variants were only transiently transfected, and the\ntransfection efficiency was usually around 40%. Therefore, some cells are positive for SEPT2-\neGFP, but negative for mCherry-tagged PIPKI.\n- Figure 2.f: Did the authors perform the same experiment testing for the i3 isoform? Did they\nobtain the same phenotype as for i5 mutant?\nWe did not perform the same experiment with the i3 isoform. But as both isoforms co-localize\nwith actin filaments, we expect a similar outcome. Also, in most cases we show that re-\nintroduction of PIPKI-i3 is sufficient to recue defects.\n- Supplementary Fig.3: The numbering for the different panels differs from the font/numbering\nused for all other figures. This should be homogenized.\nWe apologize for this inconsistency and adapted font and numbering.\n- Page 9, top (l 246-253): The described result is not displayed or it is unclear?\nWe apologize for this unclarity. The results are displayed in Figure 3a, as indicated in former\nline 246. We now added an additional reference to this figure panel in line 259.\n- Figure 3.d: On the siOCRL panel, the signal from citron kinase seems much higher than in\nthe panels above and below. However the quantification (3.f) indicates a rather constant\nintensity depending on the conditions. Can the authors comment or correct accordingly?\nThe quantification depicted in Figure 3f provides an average of 4 independent experiments,\nwith CIT and PIP2 levels quantified from at least 15 cytokinetic cells in each experiment. Please\nnote that for confocal image acquisition the focus was set on the midbody in the center of the\nICB. In some cases this layer coincides well with the ventral surface of the cells (as in the case\nof the siOCRL panel depicted in Figure 3D). In most other cases, however, the midbody plane\nlocates significantly above the dorsal surface of the cell, and the image does not appropriately\nreflect protein abundance in the cytoplasm.\nThe relative CIT-K intensity level determined from the siOCRL panel depicted in Figure 3D was\n0.88, thus, a bit lower than average determined from siControl cells.\nTo avoid confusion we now replaced the siOCRL panel with a panel that more closely\nresembles average total CIT-K levels.\n- Supplementary fig. 4.g: The septin pattern observed for i5 WT looks different from others and\nseptins seem to localize in the midbody as well. Can the authors comment on this?\nWe thank the reviewer for his critical alertness. All depicted figure panels represent snapshots\nof cells fixed during cytokinesis at slightly differing stages. As reported by Renshaw septins\ntransiently colocalize with anillin at the flanks of the midbody, before they redistribute along the\nICB. We thus assume that the septin staining depicted for i5 WT corresponds to this transition\nstate. To avoid confusion we replaced the figure panels presented for i5 WT for a more\nconsistent example.\n- Figure 5.k: The signals for MKLP1 and septins from an IP via PIPKIɣ are rather weak. Can\nthe authors comment on this? Testing for centralspindlin, have the authors also assayed\nRacGap1?\nThe signal for MKLP1 gained from an IP via PIPKI are indeed weak, but we observe a\nsignificant enrichment of MKLP1 when compared to the signal from Input lanes. We found also\nRacGAP1 to co-immunoprecipitate with PIPK1 in a different experiment (exp. 21-03-25;\nplease see figure panel below).\nThe septin to co-purifiaction with PIPKI might be impaired sterically by the antibody used for\naffinity purification of PIPKI (raised against the C-terminal tail).\nCentralspindlin affinity-purifies together with\nPIPKI.\nEndogenous PIPKI was immunoprecipitated from\nlysates of synchronized HeLa cells. The affinity-\npurified material was separated by SDS-PAGE and\nanalyzed by Western blotting using the indicated\nantibodies.\nReviewer #2\nThe manuscript submitted by Russo and colleagues addresses the question of how PIP2 is\ngenerated at the intercellular bridge during mammalian cytokinesis and what effect it has on\ncytokinetic progression. They report that coupling of the PI-5-P kinase PIPKIγ with septins is\nrequired to stabilize the centralspindlin complex at the midbody/Flemming body in late\ntelophase. This study addresses an important and unresolved question in the field and is thus\nof significant interest to cell division researchers. Additionally, it reveals an interesting coupling\nof the septin cytoskeleton to lipid synthesis. However, a number of clarifications are required.\nWe thank the reviewer for appreciating the significance of our study.\nMajor points:\n1. A major deficiency is the manuscript are a lack of rescues for siRNA-mediated depletions in\nmultinucleate experiments described in Figures 1 and Supp Figure 3D. Given that the\nphenotypes are small in absolute terms, these rescues are particularly important to establish\nthat the relatively small increases observed for cytokinesis failure are not due to off-target\neffects. While the rescues shown in Fig 3i are appreciated, they are not direct evidence of a\nrescue of cytokinesis failure. Rescues would preferably be undertaken with strategies that\navoid over-expression.\nThis is a valid concern.\nWe performed rescue experiments using stably transfected cell lines expressing mCherry, or\nsiRNA-resistant, mCherry-tagged PIPKI-i5 (WT). As depicted in our novel Supplementary\nFigure 3e depletion of endogenous PIPKI-i3/i5 significantly increased the fraction of\nmultinucleated cells in mCherry-expressing cells, but not in cells expressing mCherry-tagged\nPIPKI-i5 (WT). We these data unequivocally demonstrate that cytokinesis failure is not an off-\ntarget effect of the siRNA used throughout our experiments.\n2. The phenotype induced by PIPKIγ disruption (either by siRNA alone or with expression of\nmutants) requires more defining. If PIPKI is important for telophase progression, is there a\nmeasurable delay in telophase? Does abscission take longer? Some timing experiments could\nhelp clarify\nWe agree that timing experiments are helpful to dissect between defects occurring in early or\nlate cytokinesis.\nWe used live cell imaging of cells labelled with SiR-tubulin, to follow microtubule dynamics in\ncontrol and PIPKI-i3/i5-depleted cells. This approach allowed us to measure the time from\ninitiation of mitosis (marked by cell rounding) until completion of furrow ingression (marked by\nthe appearance of a thin microtubule bridge), and thereafter until microtubule cut (marked by\na sudden retraction of the microtubule bundle from the ICB into one of the daughter cells).\nOur analyses (depicted in novel Supplementary Figure 3g) revealed no significant change\nin furrow ingression in kinase-depleted cells, but indicated a significant delay of the microtubule\ncut.\n3. Some terms used in the manuscript are unclear and ambiguous. The common one of\n‘compact’ to describe anillin localization is unclear and does not reflect what can be seen in\nthe images. (Fig 1,3). Nor does this reflect previous studies that defined anillin dynamics in the\nintercellular bridge (El Amine et al. 2013, Renshaw et al. 2014). These two studies\ncharacterized in detail anillin dynamics and define common organizational states that reflect\ndifferent phases of intercellular bridge assembly. Assessing if and/or when these previously\ndefined patterns of anillin localization are disrupted by the various perturbations performed in\nthis study will make comparisons across studies much easier.\nWe thank the reviewer for pointing this out. We now phrase less ambiguously to describe the\nlocalization of anillin (“cells with anillin at the ICB”). Furthermore, we assessed the localization\nof anillin also at earlier stages of cytokinesis. In line with our observation that PIPKI-i3/i5 have\nno obvious effect on furrow ingression time we find septins and anillin associated with the\ningressing furrow (novel Supplementary figure 3f) in control and in kinase-depleted cells.\nLikewise, active myosin as evaluated by immunostainings of phospho-myosin LC remains\nlargely unaffected by depletion of PIPKI-i3/i5 (novel Supplementary Figure 3h).\n4. Related to point 3, in many cases the micrographs being compared appear to be at different\nstages of intercellular bridge development (i.e. that the authors are comparing two ICBs of\ndiffering ages). This occurs frequently in Figs 3 , S3 and 4. Including tubulin staining to the\nexperiments would help clarify these issues as the degree of microtubule compaction has\nbeen used to assess the intercellular bridge age thereby allowing more direct “apples to apples”\ncomparisons. This is a very important point with respect to the conclusions made and should\nbe rigorously addressed.\nWe agree that under control conditions the appearance of the acetylated tubulin staining is\nindicative of the maturation state of the ICB. However, our live experiments (Movies 1 and 2),\nin which we monitored the distribution of eGFP-SEPT2 in genome-edited cells, clearly show\nthat the ICB between the two daughter cells never elongates, irrespective of how long we wait.\nWe, therefore, feel that the appearance of acetylated tubulin bridge is not an adequate marker\nfor the maturation state of the bridge, but rather a defect caused by the abolished recruitment\nof septins onto ICB microtubules, and the concomitant destabilization of the microtubule\nbundle.\nTo more closely illustrate this phenotype we measured the width of the microtubule bundle at\na distance of 4 µm apart from the center of the ICB. Our novel Figure 4e clearly shows that\ndepletion of PIPKI-i3/i5 impairs bundling in the periphery of the ICB, and that this defect is\nrescued by introduction of siRNA-resistant PIPKI-i3 or i5 (WT), but not of kinase-dead or of\nseptin binding-deficient variants.\nMinor points:\nA. In the introduction the Piekny and Maddox review is extensively cited. While this is a good\nreview it is 15 years old and much has been learnt subsequently about anillin, who it binds to\nand how it functions. It would be useful to newcomers to the field if more up to date references\nabout specific points were used.\nWe followed the reviewer´s suggestion and updated the references for anillin functions.\nB. The biology of septins needs a more thorough treatment, again with up to date references.\nWhile it is said that septins hetero-oligomerize, the hexamer/octamer distinction is bound to be\nconfusing to non-experts and should be better developed. In addition, in the results section\n(line 170) the authors state that septins bind to the C-term of anillin. While this is the conclusion\ndrawn from this 2000 paper, subsequent work suggests a more complex relationship. This\ncombined with a better discussion of hexamers and octamers would bring the paper up to date\nand allow a subsequent more detailed discussion (and perhaps experimental plan) of the\nunderlying mechanism at play.\nWe thank the reviewer for pointing this out and also updated information about the interplay\nbetween septin and anillin.\nC. In the results, a simple statement that cells were synchronized by double thy + nocodazole\ntreatment should be added to the main text to aid the reader.\nWe included a brief statement in the Results section to aid the reader.\nD. Experiments demonstrating the localization of anillin and septins in PIPKIγ-depleted cells\nacross the stages of cytokinesis (as shown for control cells in Fig 3a) would be helpful. As the\nauthors state, anillin and septin recruitment to the equatorial membrane prior to furrow\ningression are known to be dependent on PI(4,5)P2. However, the manuscript only shows\ndisrupted telophase localization of anillin in knockdown/mutant conditions. This may result from\ndeficiencies in recruitment to the furrow/ICB in anaphase-early telophase as opposed to the\naction of PIPKIγ later in telophase.\nThis relates to the concern raised in point 3. As indicated above we find septins and anillin\nassociated with the ingressing furrow (novel Supplementary figure 3f) in control and in\nkinase-depleted cells. Likewise, active myosin as evaluated by immunostainings of phosphor-\nmyosin LC remains largely unaffected by depletion of PIPKI-i3/i5 (novel Supplementary\nFigure 3h). We conclude that the interplay between septins and PIPKI-i3/i5 does not affect\nanaphase-to-telophase transition, but rather affects ICB morphology and stability during late\ncytokinesis. But we note, that we cannot rule out additional effects of other isoforms of PIPKI.\nThese would need to be addressed in future studies.\nE. What effect does knockdown of PIPKIα & β have on PI(4,5)P2 levels in anaphase and\ntelophase cells? The results of Fig 3 would suggest that PIPKIγ is responsible for the majority\nof PI(4,5)P2 present in cytokinetic cells. Is the remaining PI(4,5)P2 is sufficient to promote\ninitial anillin/septin ICB recruitment?\nBased on the observation that loss of PIPKI-i3/i5 does not impact on anillin (and concomitant\nseptin) recruitment to the early ICB we conclude that other type I PIPK enzymes (PIPKI and/or\nPIPKI and/or other isoforms of PIPKI ) provide a sufficiently large pool of PI(4,5)P ) to allow\n2\nfor unimpaired furrow ingression.\nF. Fig 4: Measurements comparing the width of ICB bridges across conditions should be\nperformed. If as the authors argue, depletion of PIPIKIγi3-5 perturbs the development of the\nICB such that its microtubules do not compact as much as control, then the width of acetylated\ntubulin staining will better bolster this point than measurements of ICB length.\nThis relates to major concern 4.\nWe measured the width of the microtubule bundle in the center of ICB, and at a distance of 4\nµm apart. Our novel Figure 4e clearly shows that depletion of PIPKI-i3/i5 impairs bundling in\nthe periphery of the ICB, and that this defect is rescued by introduction of siRNA-resistant\nPIPKI-i3 or i5 (WT), but not of kinase-dead or of septin binding-deficient variants. Please note\nthat the acetylated tubulin staining needed to be boosted to visualize “fluffy” MTs for\nquantifications, which are not so apparent in the figure panels presented throughout the\nmanuscript.\nG. Fig5hi: I do not think it’s appropriate to measure fluorescence intensities across different\nexpanded samples unless steps were taken to ensure some normalization of expansion factors\nand a comparison to some constant. The obvious differences in intensity presented in panel g\nare more than sufficient to support the authors’ claims.\nAs indicated in the Methods section we did calculate expansion factors to ensure comparability\nbetween different conditions and specimen. Yet, we agree that the differences in intensity\ndepicted are obvious, and we removed the quantification.\nH. In S3G: the i5ΔsupSB panel contains a cell that appears to be at an earlier stage of\ncytokinesis compared to the other cells in this panel (based on less compaction of tubulin\nstaining). It further also looks to be multinucleated. Is this the norm? If yes this would have\nimplications in interpretation.\nWe assume that the reviewer refers to Supplementary Figure 4g. As delineated in our response\nto point 4 we would like to stress again that the defective ICB is part of the phenotype. None\nof the stably transfected cell lines expressing mCherry or mCherry-tagged kinase variants\ndisplays elevated levels multinucleation (see also quantification in novel Supplementary\nFigure 3e). Hence, to better represent our data we exchanged multinucleated cells in\nSupplementary Figure 4g (i5ΔSB) and in Supplementary Figure 3j (i5 K188A) and (i5ΔSB).\nI. line 76 PI(4,5)2 should be PI(4,5)P2\nWe thank the reviewer for carefully reading the manuscript. We corrected accordingly.\nReviewer #3\nThis manuscript studies the spatiotemporal control of PI(4,5)P2 during cytokinesis and reports\na key role for two splice isoforms of PIPKIgamma. The authors demonstrate that PIPKIg is\nrequired for cytokinesis progression and for the organization of anillin and septins at the\nintracellular bridge (Figure 1). They furthermore identify two splice isoforms (i3 and i5) of\nPIPKIg that can interact with septins (Figure 2)and demonstrate that these are cricital for\nproper anillin accumulation and PI(4,5)P2 synthesis near the midbody (Figure 3), as well as\nfor the recruitment of septin to the intracellular bridge (Figure 4) and the association of the\ncentralspindlin complex tot the midbody (Figure 5). Based on these results, the authors\npropose a model in which septins recruit PIPKg to the cleavage furrow and generates local\nPI(4,5)P2, whereas later on septin-bound PIPKg create even more local PI(4,5)P2 that\ncontributes to septin retention and subsequent microtubule stabilization. Overall, the data is\nconvincing and the writing is clear. Nonetheless, I do have a number of comments that need\nto be properly addressed to make the manuscript ready for publication.\nWe thank the reviewer for appreciating the validity of our data.\n1. I found the authors conclusions on the interdependence of septins and PIPKIg unclear.\nInitially, the authors postulate that septin recruits PIPKIg to the midzone, whereas later on they\nsuggest that PIPKIg is key for proper localization of SEPT6, including its localization to\nmicrotubules. The text and cartoons should be improved to clarify how the authors think about\nthis interdependence.\nAs indicated in our response to comment 1 of reviewer 1 we assessed the distribution of\nmCherry-tagged PIPKI-i5 (WT) under different knockdown conditions, and indeed find that\nkinase recruitment to the late stage ICB is impaired upon depletion of SEPT7 (a septin paralog\nessential for the assembly of septins into filaments (novel Supplementary Figure 5d).\nLikewise, mCherry-PIPKI-i5 (SB) is not properly recruited to the ICB (Supplementary Figures\n3j and 4g). Based on these findings we conclude that septins are required for localizing septin\nbinding PIPKI isoforms to the ICB. We modified cartoons and text and hope this\ninterdependence is now clearer.\nAs anillin/septin recruitment to the cleavage furrow and furrow ingression time (novel\nSupplementary Figures 3f/g) remained unaffected by kinase depletion, we did not analyze\nin depth the early-stage localization of PIPKI at the cleavage furrow. Yet, we do see coincident\nrecruitment of septins and mCherry-PIPKI-i5 (WT) to the anillin-enriched cleavage furrow, and\nre-phrased accordingly.\n2. In addition, the whole interplay between septins, PIPKIg and microtubule stabilization is not\nwell developed and therefore not very convincing. How does production of PI(4,5)P2 helps to\nenrich septins on microtubules?\nNovel data depicted in updated Figure 5j and novel Supplementary Figure 5e demonstrate\nthat septins form an endogenous complex with PRC1 and localize to eGFP-PRC1-induced\nmicrotubule bundles in transfected fibroblasts. Our data indicate that septins at the ingressing\ncleavage furrow form complex with centralspindlin as soon as the furrow approaches the\nmicrotubule bundle at the center of the ICB. Septin-bound PIPKI-isoforms also associate with\ncentralspindlin, and this initiates PI(4,5)P synthesis at the midbody (maybe fostered by\n2\ncentralspindlin-bound Arf6, a known activator of PIPKI, Krauss et al., 2003, PMID 12847086)\nto stably tether centralspindlin to the plasma membrane. Upon completion of furrow ingression\nseptins are released from the plasma membrane by an unknown mechanism, and instead\nassociate with centralspindlin-/PRC1-positive microtubules. Upon completion of furrow ingression\nseptins are released from the plasma membrane by an unknown mechanism, and associate\nwith centralspindlin-/PRC1-positive microtubules. Prescence of septins and PRC1 may further\nfavor their bundling.\nIn absence of PIPKI-i3/i5 PI(4,5)P levels at the midbody are too low to stably anchor\n2\ncentralspindlin, leading to impaired microtubule bundling along the ICB, and a loss of PRC1.\nTherefore, upon release from the plasma membrane septins are not retained on microtubules\nbut rather accumulate at PI(4,5)P -enriched sites in the periphery of the ICB.\nupdated the Discussion.\n3. The title and discussion put a lot of emphasis on nanoscale synthesis, but it is unclear if this\nmeans anything else than localized, restricted synthesis. Of course, if an individual protein\nmodifies lipids this can always be considered nanoscale synthesis, but the current formulations\nare raising the expectations that the authors have discovered nanosized membrane regions\nthat carry specific modification. This is not at all reflected in the presented data.\nWe agree, and rephrased.\nMinor comments\n4. Figure S3 – panel labels are inconsistent with all other figures\nWe thank the reviewer for his awareness. We corrected the panel labels.\n5. Figure 2f – The merge images of SEPT2 and PIPKg are confusing and would be more clear\nif presented as separate images. The green is not clear in the WT situation.\nWe included separate images in the figure panels to more clearly indicate the localization of\nSEPT2 and PIPKI.\nDetailed response to the referees\nWe would like to thank the editor as well as all referees once again for their careful reading of\nour manuscript and for their thoughtful suggestions. We were very happy to learn that all three\nreviewers and the editor agree on the high general interest and timeliness of our study and we\nwould like to thank them for their strong support to publish our findings in Nature\nCommunications. We have now made textual changes to our revised manuscript that fully\naddress all questions and concerns raised in the second round of review as detailed in our\nresponse below (in blue).\nReviewer #1:\nThe authors have performed a significant amount of additional experiments to answer the\nreferee's comments and modified the manuscript accordingly. I would thus recommend its\npublication in Nature communications in its current form.\nWe thank the reviewer for his positive decision!\nReviewer #2\nThe authors have made multiple changes to the manuscript to improve it and have addressed\nthe points I raised in the first review.\nWe thank the reviewer for appreciating the improvements of our manuscript.\nI have a few minor comments that when rectified will help the manuscript.\n1. On line 151 I think it should be sup fig 1f,g not 1e,f.\nWe apologize for this mistake, and corrected accordingly.\n2. On and around line 180 the authors cite a JCB paper from Estey et al to support their\nstatement that septins form a diffusion barrier to restrict the spread of cytokinetic proteins in\nthe intercellular bridge. Re-reading that paper I find that the Estey paper does not demonstrate\nthat nor state that nor really discuss that concept. However, Estey et al in 2011 in a review in\nCur. Biol. do discuss that concept based on findings in yeast and at the base of cilia. However,\nI am unaware of any experimental evidence to show that septins acts as a diffusion barrier in\nthe intercellular bridge. I would recommend re-wording that section significantly and toning\ndown that model unless there are other studies that demonstrate the concept of a diffusion\nbarrier, which if there are they should be cited and I apologize.\nIn their study Estey et al. demonstrate that SEPT9 is required for exocyst deposition at the\nmidbody, where the two are found adjacent to each other, rather than being colocalized. We\nagree that the authors do not provide direct evidence that septins act as a diffusion barrier at\nthe ICB. We, thus, re-phrased as follows (lines 188-191):\nAlthough largely dispensable during furrow ingression in mammalian cells, septins exert pivotal\nfunctions once the ICB is established and extends by compartmentalizing select cytokinetic\nproteins at the midbody and by scaffolding the assembly of the abscission machinery.\n3. Around lines 280-283 the authors state no effect of the treatment on the initial recruitment\nof anillin and septins to the furrow and cite their Sup Fig 3f. However, that figure whilst showing\nanillin and septins in the same cellular location +/- treatment, show cells where furrow\ningression will have stopped and are about to transition into intercellular bridge assembly and\nelongation. I have no doubt based on the figure their statement is correct, but the figure per se\ndoes not directly support their statement. A figure with images showing a furrow would support\ntheir statement.\nWe thank the reviewer for pointing this out. We added an additional panel depicting anillin and\nseptin colocalization at the cleavage furrow also at early telophase, and demonstrate that this\nis independent of presence or absence of PIPKI-i3/i5 (please see modified Supplementary\nFigure 3f).\n4. There is a problem with panel labeling in fig 5. The text refers to panels j and k which are\nnot present but are presumably h and i.\nWe apologize for the mistake, and corrected figure numbering and the legend accordingly.\n5. In sup fig 5d there is a ghost grey box that should be removed.\nWe did not find a ghost grey box in Supplementary Figure 5d, but in Figure 2f and in\nSupplementary 2d, which we removed.\nReviewer #3\nIn their rebuttal, the authors refer to a new Figure 5j. However, the revised manuscript does\nnot have a Figure 5j, despite the legends going to all the way to k. Before spending my time\non reviewing this revision, it would be great to have a proper version.\nWe deeply apologize for the inaccurate figure numbering, which is now corrected in the revised\nversion of our manuscript.\nWe include once again our response to this reviewer´s initial comments:\nThis manuscript studies the spatiotemporal control of PI(4,5)P2 during cytokinesis and reports\na key role for two splice isoforms of PIPKIgamma. The authors demonstrate that PIPKIg is\nrequired for cytokinesis progression and for the organization of anillin and septins at the\nintracellular bridge (Figure 1). They furthermore identify two splice isoforms (i3 and i5) of\nPIPKIg that can interact with septins (Figure 2)and demonstrate that these are cricital for\nproper anillin accumulation and PI(4,5)P2 synthesis near the midbody (Figure 3), as well as\nfor the recruitment of septin to the intracellular bridge (Figure 4) and the association of the\ncentralspindlin complex tot the midbody (Figure 5). Based on these results, the authors\npropose a model in which septins recruit PIPKg to the cleavage furrow and generates local\nPI(4,5)P2, whereas later on septin-bound PIPKg create even more local PI(4,5)P2 that\ncontributes to septin retention and subsequent microtubule stabilization. Overall, the data is\nconvincing and the writing is clear. Nonetheless, I do have a number of comments that need\nto be properly addressed to make the manuscript ready for publication.\nWe thank the reviewer for appreciating the validity of our data.\n1. I found the authors conclusions on the interdependence of septins and PIPKIg unclear.\nInitially, the authors postulate that septin recruits PIPKIg to the midzone, whereas later on they\nsuggest that PIPKIg is key for proper localization of SEPT6, including its localization to\nmicrotubules. The text and cartoons should be improved to clarify how the authors think about\nthis interdependence.\nAs indicated in our response to comment 1 of reviewer 1 we assessed the distribution of\nmCherry-tagged PIPKI-i5 (WT) under different knockdown conditions, and indeed find that\nkinase recruitment to the late stage ICB is impaired upon depletion of SEPT7 (a septin paralog\nessential for the assembly of septins into filaments (novel Supplementary Figure 5e).\nLikewise, mCherry-PIPKI-i5 (SB) is not properly recruited to the ICB (Supplementary Figures\n3k and 4g). Based on these findings we conclude that septins are required for localizing septin\nbinding PIPKI isoforms to the ICB. Later, PIPKI-i3/i5-dependent synthesis of PI(4,5)P is\n2\nrequired to facilitate centralspindlin- and PRC1-dependent translocation of septins onto\nmicrotubules. Specifically, centralspindlin, by tethering bridge microtubules to the furrow\nmembrane, may bring septins into close proximity with the cytokinetic bridge and thereby\nfacilitate their translocation. PRC1, on the other hand, may assist septin translocation onto\nbridge microtubules by directly interacting with septins (please also see our response to point\n2 for further clarification). We modified cartoons and text and hope our view of this\ninterdependence becomes now clearer.\nAs anillin/septin recruitment to the cleavage furrow and furrow ingression time (novel\nSupplementary Figures 3f/g) remained unaffected by kinase depletion, we did not analyze\nin depth the early-stage localization of PIPKI at the cleavage furrow. Yet, we do see coincident\nrecruitment of septins and mCherry-PIPKI-i5 (WT) to the anillin-enriched cleavage furrow, and\nre-phrased accordingly.\n2. In addition, the whole interplay between septins, PIPKIg and microtubule stabilization is not\nwell developed and therefore not very convincing. How does production of PI(4,5)P2 helps to\nenrich septins on microtubules?\nNovel data depicted in updated Figure 5h and novel Supplementary Figure 5c demonstrate\nthat septins form an endogenous complex with PRC1 and localize to eGFP-PRC1-induced\nmicrotubule bundles in transfected HeLa cells. Our data indicate that septins at the ingressing\ncleavage furrow form complex with centralspindlin as soon as the furrow approaches the\nmicrotubule bundle at the center of the ICB. Septin-bound PIPKI-isoforms also associate with\ncentralspindlin, and this initiates PI(4,5)P synthesis at the midbody (maybe fostered by\n2\ncentralspindlin-bound Arf6, a known activator of PIPKI, Krauss et al., 2003, PMID 12847086)\nto stably tether centralspindlin to the plasma membrane. Upon completion of furrow ingression\nseptins are released from the plasma membrane by an unknown mechanism, and associate\nwith centralspindlin-/PRC1-positive microtubules. Prescence of septins and PRC1 may further\nfavor their bundling.\nIn absence of PIPKI-i3/i5 the retention of centralspindlin at the midbody is impaired, as is the\ntranslocation of septins onto microtubules. Of note, PRC1 levels decline concomitantly, which\nmay reflect a mutual stabilization of septins and PRC1 on bundled microtubules. As a\nconsequence, uppon release from the plasma membrane septins are not retained on\nmicrotubules, but rather accumulate at PI(4,5)P -enriched sites in the periphery of the ICB.\nupdated the Discussion.\n3. The title and discussion put a lot of emphasis on nanoscale synthesis, but it is unclear if this\nmeans anything else than localized, restricted synthesis. Of course, if an individual protein\nmodifies lipids this can always be considered nanoscale synthesis, but the current formulations\nare raising the expectations that the authors have discovered nanosized membrane regions\nthat carry specific modification. This is not at all reflected in the presented data.\nWe agree, and rephrased.\nMinor comments\n4. Figure S3 – panel labels are inconsistent with all other figures\nWe thank the reviewer for his awareness. We corrected the panel labels.\n5. Figure 2f – The merge images of SEPT2 and PIPKg are confusing and would be more clear\nif presented as separate images. The green is not clear in the WT situation.\nWe included separate images in the figure panels as Supplementary Figure 2d to more clearly\nindicate the localization of SEPT2 and PIPKI."}, {"round": 2, "reviewer_comments": {"reviewer_1": "The authors have performed a significant amount of additional experiments to answer the referee's comments and modified the manuscript accordingly.\nI would thus recommand its publication in Nature communications in its current form.", "reviewer_2": "The authors have made multiple changes to the manuscript to improve it and have addressed the points I raised in the first review.\nI have a few minor comments that when rectified will help the manuscript.\n1. On line 151 I think it should be sup fig 1f,g not 1e,f\n2. On and around line 180 the authors cite a JCB paper from Estey et al to support their statement that septins form a diffusion barrier to restrict the spread of cytokinetic proteins in the intercellular bridge. Re-reading that paper I find that the Estey paper does not demonstrate that nor state that nor really discuss that concept. However, Estey et al in 2011 in a review in Cur. Biol. do discuss that concept based on findings in yeast and at the base of cilia. However, I am unaware of any experimental evidence to show that septins acts as a diffusion barrier in the intercellular bridge. I would recommend re-wording that section significantly and toning down that model unless there are other studies that demonstrate the concept of a diffusion barrier, which if there are they should be cited and I apologize.\n3. Around lines 280-283 the authors state no effect of the treatment on the initial recruitment of anillin and septins to the furrow and cite their Sup Fig 3f. However, that figure whilst showing anillin and septins in the same cellular location +/- treatment, show cells where furrow ingression will have stopped and are about to transition into intercellular bridge assembly and elongation. I have no doubt based on the figure their statement is correct, but the figure per se does not directly support their statement. A figure with images showing a furrow would support their statement.\n4. There is a problem with panel labeling in fig 5. The text refers to panels j and k which are not present but are presumably h and i\n5. In sup fig 5d there is a ghost grey box that should be removed.", "reviewer_3": "In their rebuttal, the authors refer to a new Figure 5j. However, the revised manuscript does not have a Figure 5j, despite the legends going to all the way to k. Before spending my time on reviewing this revision, it would be great to have a proper version"}, "author_response": ""}, {"round": 3, "reviewer_comments": {"reviewer_2": "The authors have now satisfactorily addressed all the points I have raised.", "reviewer_3": "The authors have properly addressed my comments"}, "author_response": ""}]

Decision letter