EMBO/soda-nel huggingface dataset:
A Protein Palmitoylation Cascade Regulates Microtubule Cytoskeleton Integrity in Plasmodium (2020)
Figure 2

Figure 2. Polarized co-localization of ISP1 and ISP3 at zygote apical patch

A IFA of ISP1 and ISP3 expression from zygote to ookinete of the isp1::6HA and isp3::6HA parasites, respectively. Scale bar = 5 μm.

B Co-localization of ISP1 and ISP3 from zygote to ookinete in the doubly-tagged strain (DTS, isp1::3V5;isp3::6HA). Scale bar = 5 μm.

C Western blot of ISP1 and ISP3 expression in zygotes of isp1::6HA and isp3::6HA parasites. The numbers are the relative intensities of band in blot. Right panel: the quantification of band intensity. Values are means ± SEM (n=4 biological replicates), two-tailed t test, ****, P<0.0001.

D Western blot of ISP1 and ISP3 expression in zygotes of DTS, DTS/Δisp1, and DTS/Δisp3 parasites. The numbers are the relative intensities of band in blot. Three replicates performed.

E RT-qPCR analysis of isp1 and isp3 transcripts in zygotes of the WT, Δisp1, Δisp3, and Δisp1/3 parasites. The expression is normalized to the 18s rRNA. Values are means ± SEM (n=4 biological replicates).

F IFA analysis of ISP1 and ISP3 expression dynamic of DTS/Δisp1 and DTS/Δisp3 parasites. Scale bar = 5 μm.

G Proposed model of polarized co-localization and expression compensation between ISP1 and ISP3.
A Protein Palmitoylation Cascade Regulates Microtubule Cytoskeleton Integrity in Plasmodium (2020)
Figure 3

Figure 3. Palmitoylation is critical for ISP1/ISP3 polarization and ookinete differentiation

A Structure and N-terminal residues for potential acylation of ISP1 and ISP3.

B Fluorescent microscopy of ISP and its N-terminal 20 residues (N20) fused with a BFP::3V5 peptide episomally expressed in zygotes. Scale bar = 5 μm.

C Solubility assay detecting membrane association of ISP1_FL and ISP1_N20 using different detergents. Cytosolic soluble proteins are in hypotonic buffer (Hypo), peripheral membrane proteins in carbonate buffer (Carb)，integral membrane proteins in Triton X-100 buffer (Trx), and insoluble proteins in pellet (P). BFP::3V5 is in the soluble fraction, while ISP1_FL and ISP1_N20 are in the membrane-associated fractions. ER protein BiP is a loading control.

D Acyl-RAC method detecting palmitoylation of ISP1 and ISP3 in zygotes of the DTS strain. Proteins from F (flow-through), W (wash), and E (elution) are analyzed. P28 as a loading control.

E Substitutions of N-terminal cysteine or glycine residues to alanine (G2A or C7A/C8A) ablates palmitoylation of ISP1. ISP1 was fused with BFP::3V5 and episomally expressed in zygotes. Right panel shows quantification of palmitoylated protein band from two independent repeats, values are means ± SD.

F G2A and C7AC8A mutations cause cytoplasmic distribution of ISP1 during zygote to ookinete differentiation. Scale bar = 5 μm.

G Membrane association of ISP1 with either G2A or C7AC8A substitutions.

H Expression and palmitoylation of ISP1 in ∆isp1/3 parasites complemented with the 3V5 tagged WT ISP1 or C7AC8A mutant.

I IFA of ISP1 at zygote of the complemented parasites. Scale bar = 5 μm.

J In vitro differentiation to mature ookinete of the complemented parasites. Values are means ± SEM (n=4 replicates), two-tailed t test, ***, P<0.001, ns: not significant.

K A model illustrating palmitoylation critical for apical localization of ISP1 and ISP3 at zygote.
A Protein Palmitoylation Cascade Regulates Microtubule Cytoskeleton Integrity in Plasmodium (2020)
Figure 4

Figure 4. DHHC2 directs ISP1/3 palmitoylation and ookinete differentiation

A Two-colored IFA of ISP1/DHHC2 and ISP3/DHHC2 at zygotes of the triple tagged strain isp1::3V5/isp3::6HA/dhhc2::4Myc (TTS). Right graph shows overlay of fluorescence peaks along cell periphery. Scale bar = 5 μm.

B Western blot of DHHC2, ISP1, and P28 (plasma membrane protein) of the TTS zygotes treated with PBS, trypsin (Try), or heat-inactivated (HI) trypsin. Upper panel shows the PPM and IMC structures.

C Knockdown of dhhc2 expression. Upper panel shows the promoter swap strategy applied in TTS strain, generating the dhhc2kd mutant with endogenous dhhc2 promoter replaced with clag promoter. Western blot confirming decreased expression of DHHC2 in dhhc2kd zygotes. Three replicates performed.

D Two-colored IFA of DHHC2 and P28 proteins in the TTS and dhhc2kd zygotes. Scale bar = 5 μm. Bottom panel, quantifications of DHHC2 fluorescence. Values are means ± SD (n: the number of cells analyzed). Mann-Whitney test, ****, P<0.0001.

E Western blot of DHHC2, ISP1 and ISP3 in the TTS and dhhc2kd zygotes. Two replicates performed.

F Palmitoylation of ISP1 and ISP3 in the TTS and dhhc2kd zygotes using Acyl-RAC method. NH: NH2OH. Right panel indicates quantification of protein band intensity from three replicates, values are means ± SD.

G IFA of ISP1 and ISP3 in zygotes of TTS and dhhc2kd. Scale bar = 5 μm.

H Giemsa staining of zygote/ookinete from in vitro cultured TTS and dhhc2kd parasites. Scale bar = 5 μm.

I Time-course analysis of in vitro ookinete differentiation. Values are means ± SD from three independent tests.

J IFA of DHHC2 expression in the dhhc2::6HA and dhhc2::6HA;∆isp1/3 zygotes. Scale bar = 5 μm.

K Western blot of DHHC2 in zygotes of dhhc2::6HA and dhhc2::6HA;∆isp1/3.

L IFA of DHHC2 and ISP1 in zygotes of dhhc2kd parasites complemented with 4Myc tagged WT PyDHHC2 of P. yoelii, PfDHHC2 of P. falciparum, or PyDHHC2 with PAT catalytic deficient mutation C128A. Scale bar = 5 μm.

M Western blot of DHHC2 in the complemented dhhc2kd parasites as indicated.

N In vitro differentiation to mature ookinetes of the complemented parasites. Values are means ±SD from three independent tests. two-tailed t test, ns: not significant.

O Model of DHHC2-mediated palmitoylation of ISP1 and ISP3 in zygote.
A Protein Palmitoylation Cascade Regulates Microtubule Cytoskeleton Integrity in Plasmodium (2020)
Figure 5

Figure 5. Cysteine palmitoylation in cytosolic C-terminus, catalyzed by its own PAT activity, directs IMC targeting of DHHC2

A Topology and cytosolic C-terminus (210-284 residues) of DHHC2. Lower panel indicates the conserved cysteines (residues 255, 258, 260, and 262) of DHHC2 from six Plasmodium species.

B Western blot of episomally expressed DHHC2::6HA or variants containing truncated segment (C1:210-232, C2:233-262, C3:263-284 residues) in WT zygotes.

C IFA of episomally expressed DHHC2::6HA and three truncation mutants in zygotes and retorts. Scale bar = 5 μm.

D Acyl-RAC method detecting palmitoylation of endogenous DHHC2 in the dhhc2::6HA zygotes.

E Palmitoylation of DHHC2::6HA and C4A mutant episomally expressed in zygotes. C4A: substitutions of C-terminal four cysteine to alanine. Right panel indicates the quantification of protein band intensity from two replicates, values are means ± SD.

F Cytoplasmic distribution of C4A mutant during zygote to ookinete differentiation. Scale bar = 5 μm.

G IFA of DHHC2 at the dhhc2::6HA zygotes treated with 100 μΜ 2-BP. Scale bar = 5 μm.

H In vitro ookinete differentiation of the dhhc2kd parasites complemented with either WT DHHC2 or C4A mutant. Values are means ± SD from three independent tests.

I Palmitoylation of the episomally expressed DHHC2::6HA with mutation in respective individual cysteine (C255A, C258A, C260A, and C262A). Representative of three independent repeats.

J Palmitoylation of HA-tagged and human codon optimized DHHC2 ectopically expressed in human HEK293T cells. Both C4A and C128A mutations ablate DHHC2 palmitoylation.

K DHHC2_C128A-HA mutant protein could be palmitoylated by the co-transfected DHHC2WT-Myc, but not DHHC2_C128A-Myc in HEK293T cells. Representative of two independent repeats.

L Co-immunoprecipitation of the DHHC2_WT-HA and DHHC2_WT-Myc co-expressed in HEK293T cells.

M C-terminus 75 residues peptide (Cter75) of DHHC2 could be palmitoylated as an independent substrate by co-transfected DHHC2WT-HA in HEK293T cells. Cter75 is fused with a BFP::Myc for easy detection via western blot. Cter75* peptide contains C4A mutation.

N Palmitoylation of tagged DHHC2 ectopically expressed in bacteria E.coli.

O Palmitoylation of DHHC2 ectopically expressed in bacteria treated with 2-BP.

P Model showing self-palmitoylation of DHHC2 is catalyzed by its own PAT activity, which directs proteins for IMC targeting at zygotes.
A Protein Palmitoylation Cascade Regulates Microtubule Cytoskeleton Integrity in Plasmodium (2020)
Figure 6

Figure 6. Newly assembled IMC is a polarity patch for DHHC2 targeting in fertilized zygote

A Schematic of IMC assembly during gametocyte to ookinete. IMC assembly initiates in apical and extends along protrusion of the fertilized zygotes.

B Endogenous DHHC2 is palmitoylated in the dhhc2::6HA gametocytes.

C Episomally expressed DHHC2 driven by promoter of ccp2 gene is palmitoylated in female gametocytes, female gametes, and zygotes of WT parasites. Left panel shows the expression of HA-tagged DHHC2 in female gametocyte by co-staining with antibodies of HA and α-Tubulin II (male gametocyte specific). Scale bar = 5 μm.

D IFA of DHHC2 and P28 in female gametocytes, female gametes, and zygotes of the dhhc2::6HA parasite. Gametocytes were stimulated with XA and 22°C in vitro. P28 is expressed specifically in female gametes and zygotes. Scale bar = 5 μm.

E Quantification of parasites with DHHC2 polarization in P28 positive cells during gametocyte to zygote differentiation in D. x/y: the number of cells with DHHC2 polarization and the number of cells analyzed. Values are means ±SD from three independent tests.

F No polarization of DHHC2 in the dhhc2::6HA gametocytes with only either XA or 22°C stimulation. Three replicates performed, values are means ± SD.

G No change in DHHC2 expression in zygotes of the dhhc2::6HA parasite-derived mutants with individual gene disruption of cdpk4, hap2, and dozi.

H Quantification of parasite with DHHC2 polarization in P28 positive cells. Parasites are indicated in (G) or from a genetic cross (dhhc2::6HA/cdpk4 and WT). x/y: the number of cells with DHHC2 polarization and the number of cells analyzed. Values are means ± SD from three tests, two-tailed t test, ***, P<0.001, ****, P<0.0001, ns, not significant. Cell images in the Appendix Figure S4.

I Expression and localization of DHHC2 and GAP45 during female gamete to ookinete development of dhhc2::6HA parasite. GAP45 is an IMC marker and is only detected in zygote after fertilization, but not gametes. Scale bar = 5 μm.
A Protein Palmitoylation Cascade Regulates Microtubule Cytoskeleton Integrity in Plasmodium (2020)
Figure 7

Figure 7. ISP1 and ISP3 maintain the dome-like SPM structure

A Transmission electron microscopy (TEM) cross sections of WT, dhhc2kd and ∆isp1/3 parasites showing the arrangement of SPM underneath IMC. Approximately 60 recognizable hollow microtubules (green) are associated with IMC (red), distributing evenly around circumference with an interval of 100-120 nm in WT ookinetes. Microtubules lost association with IMC in the ookinetes of dhhc2kd and ∆isp1/3 parasites. Left: one representative cell in each parasite is shown; Right: two areas are magnified to show the details. Lower panel is the quantification of distance between adjacent microtubules associated with IMC. Values are mean ± SD for n=204 (WT), 189 (dhhc2kd), and 211 (∆isp1/3) measurements in 20 cells each group from two independent experiments; Kolmogorov-Smirnov test, ****, P<0.0001.

B Apical polar ring and the emanating SPM in WT, dhhc2kd and ∆isp1/3 parasites examined by negative staining and TEM. A dome-like SPM structure radiating from the apical polar ring was observed in early (3 hours) or mature (12 hours) WT ookinetes, but impaired in mutant parasites. n is the number of cells analyzed from three independent experiments. More images in the Appendix Figure S5. Scale bar = 1μm.

C SiR-tubulin staining show the SPM structures of WT, dhhc2kd and ∆isp1/3 parasites. Arrowheads point to cell apical. Scale bar = 5 μm.

D Proposed role of DHHC2/ISP1/ISP3 in maintaining the dome-like SPM structure consisting of 60 microtubules in ookinete elongation. Microtubules lost association with IMC in the ookinetes of either dhhc2kd or ∆isp1/3 parasites.
A Protein Palmitoylation Cascade Regulates Microtubule Cytoskeleton Integrity in Plasmodium (2020)
Figure 8

Figure 8. ISP1 likely interacts with β-Tubulin in ookinete

A GST-ISP1 pulls down endogenous β-Tubulin from cell lysate of WT ookinetes. Lower panel indicates the Coomassie blue stain of recombinant GST and GST-ISP1.

B Episomally expressed ISP1::HA immunoprecipitated with β-Tubulin in WT ookinetes. Episomal vector as control.

C Endogenous ISP1::3V5 immunoprecipitated with β-Tubulin in the TTS ookinetes.

D Co-localization of ISP1::HA and β-Tubulin at ookinete periphery in IFA. Scale bar = 5 μm.

E Proximity ligation assay (PLA) detecting interaction of ISP1-HA and β-Tubulin at ookinete. Episomal vector as control. Scale bar = 5 μm.

F Solubility assay detecting membrane association of α- and β-Tubulins in WT, dhhc2kd, and ∆isp1/3 parasites. GAPDH is a cytosol soluble protein, P28 is a plasma membrane integral protein.

G Quantification of α- and β-Tubulins membrane association in F. The membrane association index is defined as the ratio of Carb fraction to Hypo fraction in F, and normalized to that of WT. Values are means ± SEM from three independent tests.

H Linear correlation of membrane association index of α-Tubulin and β-Tubulin with the conversion rate to mature ookinete among WT, dhhc2kd and ∆isp1/3 parasites. Horizontal and vertical values are means ± SD.

I Membrane association of α- and β-Tubulins in WT ookinete culture after DMSO, 2-BP, or colchicine treatment.

J Quantification of α- and β-Tubulins membrane association in I. Values are means ± SEM from three independent repeats.

K Model showing ISP1/ISP3 interaction with β-Tubulin for anchoring microtubules with IMC in the elongating ookinetes.
Muscle-derived exophers promote reproductive fitness (2021)
Figure 1

Figure 1. C. elegans muscles expel cellular content via exophers.

(A) Muscular exophers contain organelles and large protein complexes. Arrows: white - exopher, blue - muscle cell, green - mitochondria, red - proteasome foci. MOM - mitochondrial outer membrane.

(B) BMW actively releases significant amounts of exopher. The image shows the middle part of the worm's body with muscles marked with dashed lines. Arrows indicate representative exophers, and the asterisk indicates the position of the vulva.

(C) The ultrastructure of the muscular exopher and its schematic view. Arrows: red - morphologically changed mitochondria inside the exopher, yellow - normal, elongated mitochondria inside the muscle cell. c - cuticle, m - muscle, e - exopher.

(D) Comparison between muscular exopher and coelomocyte. Arrows: white - exopher, blue - muscle, green- coelomocyte.

(E) Exophers are formed via a pinching-off mechanism. Arrows: white - exopher, blue - muscle cell, red - distorted muscle cell membrane during exopher formation.

(F) Production of muscular exophers is not evenly distributed across all muscle cells. The highest number of exophers is produced by the muscles adjacent to the vulva. n = 46; N = 3.

(G) Exophers may remain connected to the sending BWM cells via thin elastic tubes that allow further transfer of cellular material. Arrows: white - exopher, blue - muscle cell, red - cellular material transferred to exopher via elastic tube.

(H) Knockdown of two autophagy genes, atg-7 and lgg-1 significantly reduce the number of generated exophers. n = 91-103; N = 3.

(I) Proteostasis disruption by hsf-1 knockdown does not increase exopher production. n = 60 and 55; N = 2.

(J) Challenging proteostasis via oxidative stress but not heat stress increases exophers production. n = 30; N = 3.

Data information: Scale bars are 10 µm (A, B, D, E, G) and 1 µm (C). Data are shown as mean ± SEM; n represents the number of worms; N represents the number of experimental repeats combined into a single value; ns - not significant, ** P < 0.01, **** P < 0.0001; (F, H, I, J) Mann-Whitney test.
Muscle-derived exophers promote reproductive fitness (2021)
Figure 2

Figure 2. Exopher formation is sex-specific and fertility-dependent.

(A) The highest number of exophers is produced during the hermaphrodite reproductive period and in aging animals. Males do not produce exophers during the first days of adulthood and begin to generate a small number of exophers later in life. Starting n = 90 hermaphrodites and 150 males; N = 3.

(B) Feminized hermaphrodites of thermosensitive a fem-1 mutant strain do not produce exophers regardless of growth temperature. This phenotype can be partially rescued by mating fem-1 mutants with males. n = 10-26; N = 2.

(C) Representative images of the middle part of the worm body in panel B.

(D) Hermaphrodites sterilized via FUdR treatment produce no exophers or only a few per animal. n = 118 and 112 animals; N = 3.

(E) Males and sterile hermaphrodites (via FUdR treatment) show the formation of spherical structures in the BWM that resemble mature exophers. MOM - mitochondrial outer membrane.

Data information: Scale bars are 10 µm. Data are shown as mean ± SEM; n represents the number of worms; N represents the number of experimental repeats combined into a single value; ** P < 0.01, **** P < 0.0001; (B, D) Mann-Whitney test.
Muscle-derived exophers promote reproductive fitness (2021)
Figure 3

Figure 3. Muscular exopheresis is a non-cell autonomous process regulated by in utero developing embryos.

(A) The number of produced exophers positively correlates with the number of in utero embryos. The violet line is a linear regression line, and each orange point represents one animal. All animals were 1-3 days old; n = 54; N = 3.

(B) RNAi knockdown of genes regulating the egg laying rate and their presence in the uterus influences exopher production. n = 50-60; N = 2.

(C) Embryos retention in the uterus caused by starvation during worm's reproductive period increases exopher production. n = 81-90; N = 3.

(D) Schematic representation of the experimental setup for investigating the influence of embryo-conditioned buffer on exopher production.

(E) Exposure of young-adult worms to embryo-derived substances increases exopher production. n = 48 and 56; N = 3.

(F) Increased eggshell permeability caused by emb-27 knockdown elevates exopher production. n = 103 and 89; N = 3.

Data information: Data are shown as mean ± SEM; n represents the number of worms; N represents number of experimental repeats that were combined into a single value; ns - not significant, * P < 0.05, **** P < 0.0001; (A) Two-tailed Student's t-test, (B, C, E, F) Mann-Whitney test
Muscle-derived exophers promote reproductive fitness (2021)
Figure 4

Figure 4. Muscular exopheresis benefits offspring development.

(A) Schematic representation of the experimental setup for investigating the influence of overactive exopheresis on F0 and F1 worm generation.

(B) Exopher production does not improve muscle functionality. n = 11 - 38; N = 3.

(C, D) Animals with overactive exopheresis have reduced exploratory behaviour presented as a reduction in straight line distance travelled (C) and by representative worms traces on the plate (D). n = 11-34; N = 3.

(E) RNAi knockdown of the egg yolk precursor protein VIT-1 increases the number of muscular exophers. n = 20; N = 2.

(F) Embryos from hermaphrodite mothers that produce a high number of exophers contain more egg yolk precursor protein VIT-2. Representative images of embryos with endogenous VIT-2::GFP levels from mothers with different exopheresis activity are shown above the graph. n = 15 and 14, N = 5 and 6. Scale bar is 10 µm.

(G) RNAi knockdown of RME-2 yolk receptor abolish exopher production. n = 110-123; N = 3.

(H) Muscle-produced VIT-2 is released from muscles via exophers. The image shows the midbody of worms expressing the proteasome subunit RPN-5 tagged with RFP in BWM and VIT-2::GFP endogenous expression. Arrows: white - exopher, blue - muscle cell. Dashed lines mark embryos present in the uterus. Scale bar is 10 µm.

(I) Offspring of worms with overactive exopheresis develop faster. Y.A. - young adult stage, L4 - last larval stage. n = 317-372; N = 2.

Data information: Data are shown as mean ± SEM; n represents number of worms; N represents number of experimental repeats that were combined into a single value; * P < 0.05, ** P < 0.01, **** P < 0.0001; (E, G) Mann-Whitney test; (F) two-tailed Welch's t-test; (I) Fisher's exact test.

Expanded View Figures Legends
Biogenesis of secretory immunoglobulin M requires intermediate non-native disulfide bonds and engagement of the protein disulfide isomerase ERp44 (2021)
Figure 2

Figure 2. Formation of µs2L2-C575OX intermediates in myelomas and hybridomas.

A. Aliquots of the lysates from J558L transfectants expressing wild type µs or µsC575A chains were resolved under non-reducing conditions before or after removal of N-glycans by PNGaseF. Blots were decorated with anti-µ antibodies. Blue and red arrows point to the closed (oxidized) and open µs2L2 species respectively.

B-C. Intracellular µs2L2 species purified from the murine hybridoma B1.8 were subjected to the sequential alkylation protocol described in panel B and analyzed by mass spectrometry (panel C). In the MS/MS spectrum Y and B ions are shown in blue and red, respectively.
Biogenesis of secretory immunoglobulin M requires intermediate non-native disulfide bonds and engagement of the protein disulfide isomerase ERp44 (2021)
Figure 4

Figure 4. ERp44 attacks the non-native disulfide bond formed by two C575 within single subunits

ERp44KO HeLa cells expressing Ig-λ chains were transfected with wild type (µ), L566A or C575A mutant µs chains and Halo-ERp44, or Halo-ERp44∆RDEL, as indicated. Empty vectors (- or EV) served as controls.

A. Aliquots from the spent media (Extracellular) were blotted under non-reducing conditions and decorated with anti-µ antibodies. The blue arrow points at the faster migrating µs2L2 released by cells expressing wild type µs or L566A chains . Expression of HaloERp44 restores C575-dependent retention, and wild type µs are secreted only as polymers (black arrow). Traces of covalent HaloERp44-µ2L2 complexes (black asterisks) are detected in the media of cells expressing HaloERp44∆RDEL and µs or L566A. The slightly faster mobility of µs2L2 and µsL complexes released by cells expressing ERp44∆RDEL suggest that the latter inhibits processing of the N563 glycan, likely hindering it to the Golgi enzymes upon non-covalent binding.

B. The corresponding cell lysates are shown in this panel. The short black arrow points at the polymers formed in ERp44KO HeLa cells upon expression of wild type HaloERp44. The black asterisks indicate covalent complexes that this transgene forms with C575 containing µ2L2. Traces of intracellular µs2L2-ERp44∆RDEL complexes are visible in µs and L566A but absent from C575A expressing cells.

Data information: All membranes were decorated with anti-μ antibodies. Black roundish spots seen in the left panels are artifacts that formed during blotting procedures and signal acquisition.

C. Lysates of the ERp44KO transfectants were precipitated with NP-sepharose, resolved under non reducing conditions and the blot decorated sequentially with anti-µ, anti-ERp44 or anti-Halo. The two black asterisks point at bands corresponding to covalent complexes containing µs2L2 and endogenous or Halo-tagged ERp44.