EMBO/SourceData huggingface dataset:
Figure 5(A-D) DNA topology of the indicated minichromosomes of increasing DNA length (kb) before (26oC) and after inactivation of cohesin (35oC) in scc1-73 cells. In each case, the first 2D gel resolves the Lk topoisomers (Lk), the second 2D gel uncovers the knotted forms (Kn). Gel signals are denoted as in Fig 2. Bottom graphs compare the Pkn before and after the inactivation of cohesin (mean ± SD from three independent experiments). P-values (Student's t test): ns, p > 0.05.(E) Plot of Pkn values of minichromosomes of increasing DNA length (including YEp13) before and after inactivation of cohesin. P-values (Student's t test): **p < 0.01.
Figure 6(A-D) DNA topology of the indicated minichromosomes of increasing DNA length (kb) before (26oC) and after inactivation of Smc5/6 complex (35oC) in smc6-9 cells. In each case, the first 2D gel resolves the Lk topoisomers (Lk), the second 2D gel uncovers the knotted forms (Kn). Gel signals are denoted as in Fig 2. Bottom graphs compare the Pkn before and after the inactivation of Smc5/6 complex (mean ± SD from two independent experiments).(E) Plot of Pkn values of minichromosomes of increasing DNA length (including YEp13) before and after inactivation of Smc5/6 complex.
figf1(a,b) Multiple MBds associate with a PC3 cell (a) and a B-lymphoblast (b). Insets: MBd labelling (a) and merged differential interference contrast microscopy image with MBd labelling to show cell boundaries (b). MKLP1, MBd marker (a,b; red); CD44, membrane (a; green); DAPI, DNA (a; blue). Scale bar, 5 μm (a) and 2 μm (b).(c,d) Three-dimensional reconstructions of polarized cells in a monolayer (c) and a HeLa cell (d) show intracellular MBds. (c) ZO-1, tight junction; MKLP1, MBds. Scale bar, 2 μm. Enlargement (bottom panel) of the box in the top panel shows five MBds (arrows). (d) Wheat-germ agglutinin, plasma membrane (red); MKLP1-GFP, MBds (green); DAPI, DNA (blue). Scale bar, 5 μm.(e) Electron microscopy of an MBd in a permeabilized MCF-7 cell showing immungold labelling with MKLP1 antibodies. Inset, lower magnification of the MBd (boxed) in the cell; nucleus, right. Scale bar, 200 nm.(f) Time-lapse images during extracellular trypsin treatment of HeLa cells show retention of most MBds (MKLP1-GFP, red). Two MBds (yellow arrows) are lost on treatment, indicating digestion and/or dissociation. Time (h:min) post trypsin. Scale bar, 5 μm.(g) Two-day co-cultures of HeLa cells expressing either MKLP1-GFP (MBd marker) or cytosolic RFP. Green MBds (arrows) associated with red cells (asterisk) indicate post-mitotic transfer of MBds between cells. Scale bar, 10 μm.
figf2(a) The CETN1-GFP signal is brighter in the upper centrosome/spindle pole of a mitotic spindle. The merged differential interference contrast microscopy image with CETN1-GFP labelling at two centrosomes shows a metaphase chromosome. Insets: enlargements and semi-quantitative integrated intensity profiles of centrioles. Scale bar, 5 μm. (b) The brighter CETN1-GFP signal represents the older centrosome, as it co-stains more intensely for hCenexin1 and remains more intense throughout cell division (Supplementary Fig. S1a). Scale bar, 5 μm. Lower left, merge.(c,d) Time-lapse images show that the mitotic midbody is preferentially inherited by the daughter cell with the older centrosome in HeLa cells (c) and hESCs (d). Cells were imaged at the indicated times (h:min) from telophase by phase-contrast microscopy (c) and from metaphase by differential interference contrast microscopy (d). Middle panel of (c) and upper left panel of (d): CETN1-GFP at centrosomes; enlargements and integrated intensity profiles show that the daughter cell with the older centrosome (c, upper; d, lower) inherits the MBd (lower right images in c,d). Mitotic midbody and MBds (c,d; arrows). MKLP1, MBd marker (red); α-tubulin, mitotic midbody and cell-boundary marker (green); DAPI, DNA (blue). Scale bars, 10 μm (c,d).
figf3(a) A histological section through mouse seminiferous tubules labelled for MKLP1 shows several MKLP1+ puncta in cells of the basal layer where stem cells reside. Scale bar, 20 μm. Inset: Enlargement of the cell marked by the arrow.(b,c) Electron micrographs of mitotic midbody (b, arrow) and multiple midbody-like structures in interphase cells with similar shapes and sizes in a juxtanuclear position (c, arrows) in basal cells of mouse seminiferous tubules. N, nucleus. Scale bars, 1 μm.(d) Representative planes of a neural progenitor cell in the ventricular zone (Sox2+, bottom left panel) of an E13.5 mouse brain show that an intracellular MBd (asterisk) is associated with the ventricle-facing daughter in the asymmetrically dividing cell (top row). The bottom row emphasizes the position of paired chromosomes in a dividing anaphase cell. CD133, midbody/MBd marker (green); Na/K-ATPase, cell-border marker (red); DRAQ5, DNA (blue); DAPI, DNA. v, ventricle. Scale bar, 5 μm. Note that abscission occurs apically in these cells.(e) A histological section through a hair follicle (left, phase-contrast microscopy) stained for the stem-cell marker keratin 15 (K15) to identify the bulge region (dotted box), the stem-cell niche. DNA stain (DAPI) and the phase-contrast microscopy image show full follicle architecture. (f) Upper panels show MBd-accumulating cells in the bulge region (boxed) co-labelled with K15 and MKLP1. Enlargements (lower panels) of the boxed region highlight a cell with four MBds (asterisks). N, nucleus. Scale bar, 5 μm.(g-i) Quantitative analysis and representative images show a decrease in MBd-accumulating cells on the differentiation of pluripotent stem cells (g) to fibroblast-like cells (h), and an increase in MBd-accumulating cells after reprogramming differentiated cells (h) to induced pluripotent stem cells (i). Numbers refer to mean±s.d., n=3. MKLP1, MBds; ZO-1, tight junctions; α-tubulin, microtubules; Aurora B, midbodies. Scale bar, 10 μm.
figf4(b) Cells pulse-chased with EdU show a decrease in EdU intensity (x axis) over time (y axis), reflecting dilution of dye after cell divisions. (c,d) After a 96 h chase period, EdU levels were compared between cells with MBd numbers of >1, 1 and 0 (y axis) in HeLa (c) and SAOS-2 cells (d). In both cases, no significant differences were noted (c, P=0.2101; d, P=0.5609, one-way analysis of variance, with at least 800 cells analysed for each experiment, n=3), indicating similar cycling rates among different subpopulations of cells. (b-d) Each graph is a representative experiment. Cells analysed are shown by green points, the medians are depicted by vertical red lines, and horizontal red lines with ticks illustrate the interquartile range.
figf5(a) Depiction of FPP assay. Digitonin selectively permeabilizes the plasma membrane but not internal membranes. Proteinase K degrades cytoplasmic components but membranous compartments remain intact. Under these conditions, MKLP1-GFP-labelled MBds (blue ellipse) in the cytoplasm will be degraded whereas those inside membrane-bound compartments will not. (b) MBds in MBd-poor hRPE-1 cells are largely protected (∼90% in membranous compartments, 10 cells analysed), whereas most MBds in HeLa cells are not (∼27%, 11 cells analysed), and are thus degraded in cytoplasm. Scale bar, 5 μm.(c) Presence of MBds in lysosomes on chloroquine or E64d-pepstatin A (E64d-PepA) inhibition in hRPE-1 and HeLa cells, but not in MCF-7 and H9 hESCs. Chloroquine treatment of H9 hESCs is not included as it caused differentiation and cell death. A representative image of hRPE-1 cells inhibited by chloroquine is shown, depicting two MBds inside lysosomes. MKLP1 and LAMP2 are used as MBd (red) and lysosome (green) markers, respectively. DAPI, DNA (blue). n=100 MBds per treatment in each of the biological triplicates. Scale bar, 5 μm.(d) Percentage of MBd+ cells (MBd levels), percentage of MBds within lysosomes and percentage of cells exiting cytokinesis following synchronization. MKLP1 and LAMP2 are used as markers as in c. Note that MBds are transferred into only one of the two nascent daughter cells after abscission (Fig. 2d), so a 50% maximum will be expected for MBd+ cells. The peak of MBds transferred to cells is 3 h after plating followed by a peak of MBds entering lysosomes at 7 h.(e) Both chloroquine and E64d-PepA treatments increase the percentage of MBd+ cells in hRPE-1 cells and HeLa cells (chloroquine, P=0.0021 and P=0.0187, respectively; E64d-PepA, P=0.0022 and P=0.0043, respectively; n=3 for all experiments). In contrast, lysosomal inhibition has no detectable effect on hESCs (H1, H9) or MCF-7 cancer cells. DMSO, dimethylsulphoxide. Data are presented as mean±s.d. (c-e), except mean±s.e.m. in hESCs (e).
figf6(a) Single-plane confocal microscopy images of MBds within LC3-positive autophagosomes in MEFs expressing GFP-LC3 (left) and in hRPE-1 cells stained for endogenous LC3 (right). MBd markers: Cep55, MKLP1 or MgcRacGAP. Autophagosomes: GFP-LC3 or LC3. Note that MKLP1 (blue) and MgcRacGAP (red) are co-localized (magenta) in the autophagosome (green), indicating that MBds are sorted into autophagosomes. Scale bars, 2 μm.(b) Decreasing autophagy levels by deletion of the Atg5 gene (left) or depletion of ATG7 by siRNA (right) significantly increases the percentage of MBd+ cells (P=0.0019 and P=0.021, respectively, n=3). Immunoblots confirm loss of the Atg5-Atg12 conjugation in mutant cells and depletion of ATG7 (asterisk). GAPDH, glyceraldehyde 3-phosphate dehydrogenase.(c) Rapamycin (Rapa) and LiCl co-treatment induces autophagy and decreases the percentage of MBd+ cells (left, HeLa; P=0.0056, n=3). Immunoblots showing increased LC3-II levels confirm autophagy induction. Induction of autophagy by overexpression of Flag-tagged BECN1 reduces the percentage of MBd+ cells (right, MCF-7; P=0.0008, n=4). Ctrl, control.(d) Representative immunoblots showing high autophagy levels in normal cells and low levels in stem cells and cancer cells. Autophagic flux (autophagic activity) was measured by changes in the levels of LC3-II, in the presence or absence of lysosomal inhibitors E64d-PepA. U, uninhibited. I, inhibited. Below, the average of the percentage change in LC3-II levels after lysosomal inhibition from three experiments. α-tubulin, loading control.
figf7(a) Single-plane confocal microscopy images showing co-localization of the MBds and the autophagic receptor NBR1 in U2OS cells and p62-deleted MEFs. MBd markers: MKLP1 or Cep55. Scale bar, 2 μm.(b) The percentage of MBd+ cells is significantly increased following the depletion of NBR1 (P=0.022, n=3), but not another autophagic receptor, p62. Co-depletion of NBR1 and p62 does not further increase MBd levels over NBR1 depletion alone.(c) Deletion of the p62 gene does not affect the percentage of MBd+ cells. For b and c, immunoblots verify protein loss.(d) Co-immunoprecipitation (IP) reveals that CEP55 and NBR1 form a complex. Precipitated proteins and 5% of the input material (Input) were analysed by immunoblotting (IB) with antibodies against NBR1 or CEP55.(e-g) Overexpression of CEP55-eGFP (enhanced green fluorescent protein) increases the percentage of MBd+ cells (e; P=0.0007, n=3) and the percentage of NBR1-negative MBds (f; P=0.0568, n=3), presumably by sequestering NBR1 (red) away from MBds in cells expressing CEP55-eGFP (green) as shown in g, and consequently preventing MBd degradation. The dotted box in g is enlarged (top right panel), and the labelling of NBR1 and CEP55-eGFP (middle and bottom right panels) are also presented. DAPI, DNA (blue). Scale bar, 5 μm. The data are presented as mean±s.d. (b,c,e,f). Uncropped images of blots are shown in Supplementary Fig. S6.
figf8(a-c) Reprogramming is more efficient after MBd enrichment. Differentiated cells (dH1f) and embryonic fibroblasts (IMR90) are reprogrammed after stable expression of either NBR1 shRNA or shNT. Emerging iPSC colonies are scored on the basis of TRA-1-60 expression. Cells depleted of NBR1 to increase MBd levels show an increase in iPSC colony formation (a,b, dH1f, 3.1±0.5-fold, n=15, P=0.00035; IMR90, 3.4±0.8-fold, n=3, P=0.02; data are mean±s.e.m.) but insignificant changes in autophagic activity (c) over shNT control. (b) Representative plates with TRA-1-60-immunostained iPSC colonies. The immunoblot (c, top) and densitometry (c, bottom; percentage of autophagic flux) show representative results (n=3); α-tubulin, loading control.(d) MCF-7 side-population (SP) cells have a significantly higher percentage of MBd+ cells over the non-side-population (MP; P=0.0015, n=3; data are mean±s.d.).(e,f) MBd enrichment in cancer cells leads to increased anchorage-independent growth. MKLP1-GFP-expressing HeLa cells are separated into 'MBd-high' and 'MBd-low' subpopulations. An increase in the 'MBd high' over 'MBd low' ratio is associated with an increase in soft-agar colony formation (e). No significant difference was observed when the enrichment of the MBd-high subpopulation was less than threefold. More soft-agar colonies are formed when MBds are enriched by NBR1 depletion (NBR1 shRNA) in HeLa (f, left; P=0.0012, n=3) and mouse 134-4 cells (f, right; P=0.0086, n=3); control, shNT. Data are mean±s.d., and the colony number (e,f) is the sum of INT-violet-stained colonies from ten random fields.
Figure 1C-F. Polysome profiling of yeast cells with (C-E) or without (F) cycloheximide (CHX) treatment, immunoblots for Stm1 and Rpl3 in polysome fractions, and quantification of Stm1 distribution across the polysome profiles of cells grown under nutrient-rich condition (C, F), rapamycin treatment for 1 hour (D), or nitrogen starvation for 1 hour (E). Individual polysome profiles were fractionated on a 5-50% sucrose density gradient containing 150 mM KCl into 9 fractions, precipitated and immunoblotted for Stm1 and Rpl3. Rpl3 is used as a loading control. Each experiment was repeated at least three times.
Figure 2G. Polysome profiles of stm1∆ cells containing either full-length or N-terminally deleted or C-terminally deleted Stm1 mutants upon nitrogen starvation for 1 hour.H. Binding of full-length or deletion mutants of Stm1 across the polysome profile fractions shown in Figure 2E. 9 equal volume fractions of each polysome profile were precipitated and analyzed by immunoblot for Stm1 and Rpl3. At least two biological replicates are used for each mutant.
Figure 3A-B. In vitro kinase assay (A) and its quantitation (B) using recombinant mTORC1 and recombinant Stm1 or Stm1AAA as a substrate in the presence of 32P-γ-ATP. Immunoblots for Stm1 and Raptor are shown. Asterisk indicates the auto-phosphorylation of a mTORC1 component. INK128 is used as an inhibitor of mTOR. Minimum of three replicates are used for statistical analysis by unpaired t-test. Data are presented as the mean ± SD.C. Polysome profiles of wild-type and stm1∆ cells under nitrogen starvation for 1 hour. D. Polysome profiles of wild-type and STM1AAA cells under nitrogen starvation for 1 hour. E. Polysome profiles of wild-type and STM1EEE cells under nitrogen starvation for 1 hour. F-
Figure 4A-B. Ribosomal subunit profiles (A) and quantitation of ribosome content (B) of stm1∆, wild-type, STM1AAA, and STM1EEE cells treated with rapamycin for 24 hours. For the ribosomal subunit profile analyses, the cell extracts were treated with 50 mM EDTA and separated on 5-25% sucrose density gradients. At least five biological replicates are used for calculation of p-values from multiple unpaired t-tests are indicated in the graphs.E-G. Immunoblot of Rpl24-GFP and free GFP (E), quantification of total GFP relative to Pgk1 (F), and free GFP relative to Rpl24-GFP (G) in stm1∆, wild-type, STM1AAA, and STM1EEE cells treated with rapamycin for 24 hours. At least six biological replicates are subjected to statistical analysis using multiple unpaired t-tests. (s.e. for short exposure; l.e. for long exposure).J-K. Immunoblot (J) and the quantification (K) of Rpl3 in wild-type and stm1∆ cells without rapamycin, or with rapamycin for 24 hours along with or without bortezomib treatment for 8 hours. At least six biological replicates are subjected to statistical analysis using multiple unpaired t-tests.
Figure 5A-B. Puromycin incorporation (A) and its quantification (B) in stm1∆, wild-type, STM1AAA, and STM1EEE cells starved of nitrogen for 24 hours and restimulated with amino acids (+ N) for 10, 20, or 30 minutes. Immunoblots for puromycin, Stm1, Rpl3, and Pgk1 are shown. Relative puromycin incorporation for at least five biological replicates is subjected to multiple unpaired t-tests.D-E. Representative fluorescent microscopic images for expression of Rpl24-GFP (D) and its intensity quantification (E) in tetrads of control (RPL24GFP) and stm1∆ RPL24GFP cells (n=100, each). Experiments are performed at least three times. Statistical analysis is done by unpaired t-test.
Figure 6A-C. Polysome profile and immunoblotting of SERBP1, RPS6, and RPL7a across the polysome fractions of HEK293T cells treated with DMSO (A), INK128 (B), or harringtonine (C) for 1 hour. Polysome profiles were performed using 5-50% sucrose density gradients containing 150 mM KCl.I-J. Immunoblot for ubiquitin (I) and its quantification (J) in the supernatant and ribosomal pellet obtained from HEK293T cells treated with control sgRNA (sgCtrl) or SERBP1 sgRNA (sgSERBP1) along with INK128 treatment for 48 hours. At least four biological replicates are analyzed by unpaired t-test. Data are presented as the mean ± SD.
Figure 2C, Comparison of wild-type colonies with phytochrome and hox-deletion strains in the presence of 0.12 mM menadion (MD), 7 mM H2O2 or 2 mM tert-butyl hydroperoxide (tBooH). The strains were incubated for 5 days at 28°C in the dark.
Figure 3E, Overlay of normalized (at 705 nm) difference spectra plus a difference spectrum of the photosensory domain in the presence of the bacterial HO BphO
Figure 4B, HoxA-GFP and CitA-GFP (ca. 170 amino acids from the N-terminus of citrate synthase) were expressed over night at 30°C in minimal medium with 2% threonine and 0.2% glucose. Mitochondria were incubated with 100 μg/ml proteinase K (PK) for 20 min on ice. Crude extract (CE), supernatant 1 (S1) containing mitochondria, pellet 1 (P1), supernatant 2 (S2) and pellet 2 (P2) with mitochondria were analyzed by Western Blot using anti GFP antibodies. CitA-GFPc = cytoplasmic form; CitA-GFPi = imported version.C, D Role of the C-terminal anchor in HoxA. Microscopy was done as in A using a fluorescent microscope without the Airyscan technology. C, Expression of GFP-HoxA lacking the C-terminal anchor (∆CTA) in A. alternata. D, Expression of GFP fused to the CTA motif.
Figure 5A, BiFC (split-YFP) analysis of HOs and phytochrome. Strains (SChS29-33) were grown over night at 28°C minimal medium with 2% threonine and 0.2% glucose. MitoTracker Red CMOXRos was used for mitochondrial staining. The mitotracker signal was observed in the RFP and the split-YFP signal in the YFP channel of the fluorescent microscope. Pictures in both channels were overlaid (merge). The left pictures show the same hyphae in differential interference contrast (DIC). The scale bar represents 10 µm.C, Co-expression of the HOXs along with the photosensory domain of A. nidulans FphA (PGP) over night at 20°C. PGP was purified via the Strep-tag system and the eluate concentrated (upper panel). The lanes were loaded as follows: negative control before induction (-), 20 h after induction (20 h) crude extract (CE), supernatant (S), pellet (P), flow through (F) and eluate (E). The fractions were analyzed by Western Blot using the Anti-His antibody (lower panel).
Figure 6A, B, Biolayer interferometry analysis with Apo-PGP A and holo-PGP B. Proteins were immobilized at a streptavidin sensor tip. Free binding sites were blocked with biotin. Association and dissociated kinetics were recorded at the indicated HoxA concentrations. Kd values were calculated with the manufacturers software using a global fit for both experiment
Figure 2  (B) BW25113(ycbB, relA') and its derivatives obtained by individual deletion of endopeptidase genes. BW25113(ycbB, relA') is an abbreviated name for BW25113 ΔrelA pKT2(ycbB) pKT8(relA').
Figure 3 Growth of BW25113(ycbB, relA'), BW25113(ycbB, relA') ΔmepM, and its derivatives harboring plasmids encoding MepM, MepM H393A, and MepM ΔLytM were tested in the presence of ceftriaxone at 8 µg/ml (+ ceftriaxone) or in the absence of the drug (- ceftriaxone) in BHI agar plates supplemented with 40 µM IPTG and 1% L-arabinose for induction of ycbB and relA', respectively. The genes encoding MepM, MepM H393A, and MepM ΔLytM were inserted into the vector pHV9 under the control of the PphlF promoter, which is inducible by 2,4-diacetylphloroglucinol (DAPG). Basal level of expression of mepM under the control of PphlF was sufficient to restore ceftriaxone resistance in the absence of the inducer.
Figure 4 The mepM gene was expressed under the control of TIS1 or TIS2 (translation initiation) and of PrhaBAD (the L-rhamnose-inducible promoter of vector pHV30). Growth of BW25113(ycbB, relA') ΔmepM harboring the pHV30 derivatives was tested on BHI agar supplemented with ceftriaxone 8 µg/ml, IPTG 40 µM (induction of ycbB) and L-arabinose 1% (induction of relA'). (A) For TIS2, growth around paper disks containing 1 or 2 mg of L-rhamnose indicated that induction of the expression of mepM was required for ceftriaxone resistance. In contrast, a higher level of translation from TIS1 was sufficient for ceftriaxone resistance in the absence of the inducer. (B) The experiment was repeated with bacteria pre-exposed to L-rhamnose that were harvested at the vicinity of the disk containing 2 mg of L-rhamnose. Expression of ceftriaxone resistance remained dependent on the presence of L-rhamnose indicating that the requirement for MepM is not transient. The diameter of the growth zones is larger in panel (B) than in panel (A) as expected from the fact that bacteria in the inoculum used in (B) had been grown in the presence of the inducer and already contained MepM. In (A) growth is only possible after diffusion of L-rhamnose in the medium prior to the action of ceftriaxone. At a distance from the disk, diffusion was not sufficiently rapid to observe resistance.
Figure 5 (A) rpHPLC chromatograms of sacculi isolated from BW25113 grown in minimal medium to stationary phase and digested by lysozyme (upper panel) or by lysozyme and 5 µM MepM (lower panel). Absorbance was monitored at 205 nm (mAU, milli-absorbance unit).
Figure 6 Functional complementation of the mepM deletion in BW25113(ycbB, relA') ΔmepM was performed with the pHV30 vector or recombinant plasmids encoding each of the eight endopeptidases under the control of the PrhaBAD promoter and of the TIS1 translation initiation signal. Induction of endopeptidase (ED) genes was performed with 0.2% L-rhamnose in the presence or absence of 8 µg/ml ceftriaxone. BHI agar plates contained 40 µM IPTG and 1% L-arabinose for induction of ycbB and relA', respectively.
Figure 8 (B) The plating efficiency assay was performed with derivatives of BW25113 Δ8EDs pHV53(mepM) harboring the vector pHV7 or recombinant plasmids carrying each of the eight endopeptidase genes under the control of the ParaBAD promoter. In this assay, functional replacement of MepM is detected based on growth in media containing 0.2% or 1% L-arabinose for expression of the endopeptidase gene carried by vector pHV7, while by-passing the requirement for induction of the mepM copy of pHV53 by L-rhamnose. Complementation was observed with both concentrations of inducer for mepH, mepS, and pbpG. Overproduction of mepM encoded by the pHV7 derivative in the presence of the high dose of L-arabinose (1%) was lethal. The right panel presents the growth control performed in the presence of 0.2% L-rhamnose for induction of the mepM copy carried by pHV53.
Figure 9 (B) Functional complementation of the mepK deletion of BW25113 ΔmepK pHV63(ycbB) was performed with the pHV30 vector or recombinant plasmids encoding each of the eight endopeptidases under the control of the PrhaBAD promoter. Induction of ycbB and of endopeptidase (ED) genes was performed with 0.2% L-arabinose and 1% L-rhamnose, respectively. BHI agar plates contained chloramphenicol (20 µg/ml) to counter-select loss of pHV63(ycbB).