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The human AAA-ATPase VPS4A isoform and its co-factor VTA1 have a unique function in regulating mammalian cytokinesis abscission [1]

['Inbar Dvilansky', 'Department Of Life Sciences', 'Ben-Gurion University Of The Negev', 'Beer Sheva', 'National Institute For Biotechnology In The Negev', 'Nibn', 'Yarin Altaras', 'Nikita Kamenetsky', 'Dikla Nachmias', 'Natalie Elia']

Date: 2024-05

Mutations in the human AAA-ATPase VPS4 isoform, VPS4A, cause severe neurodevelopmental defects and congenital dyserythropoietic anemia (CDA). VPS4 is a crucial component of the endosomal sorting complex required for transport (ESCRT) system, which drives membrane remodeling in numerous cellular processes, including receptor degradation, cell division, and neural pruning. Notably, while most organisms encode for a single VPS4 gene, human cells have 2 VPS4 paralogs, namely VPS4A and VPS4B, but the functional differences between these paralogs is mostly unknown. Here, we set out to investigate the role of the human VPS4 paralogs in cytokinetic abscission using a series of knockout cell lines. We found that VPS4A and VPS4B hold both overlapping and distinct roles in abscission. VPS4A depletion resulted in a more severe abscission delay than VPS4B and was found to be involved in earlier stages of abscission. Moreover, VPS4A and a monomeric-locked VPS4A mutant bound the abscission checkpoint proteins CHMP4C and ANCHR, while VPS4B did not, indicating a regulatory role for the VPS4A isoform in abscission. Depletion of VTA1, a co-factor of VPS4, disrupted VPS4A-ANCHR interactions and accelerated abscission, suggesting that VTA1 is also involved in the abscission regulation. Our findings reveal a dual role for VPS4A in abscission, one that is canonical and can be compensated by VPS4B, and another that is regulatory and may be delivered by its monomeric form. These observations provide a potential mechanistic explanation for the neurodevelopmental defects and other related disorders reported in VPS4A-mutated patients with a fully functional VPS4B paralog.

Here, we set out to investigate the functions of the 2 VPS4 isoforms encoded in mammalian cells during cytokinetic abscission by knocking out specific VPS4 components. We found that although cells express higher levels of VPS4B compared to VPS4A, depletion of VPS4A leads to a considerably more severe abscission delay than depletion of VPS4B. Stochastic optical reconstruction microscopy (STORM) measurements of the density of the ESCRT-III protein, IST1, at the intracellular bridge pointed to the involvement of VPS4A in early abscission and of VPS4B in late abscission, supporting a differential role for VPS4 isoforms. Unexpectedly, depletion of the VPS4 co-factor, VTA1, which stabilizes the active hexameric form of both VPS4 isoforms, accelerated abscission. Moreover, in WT cells, VTA1 was found to interact with both CHMP4C and ANCHR, suggesting its role in the abscission checkpoint. VPS4A and a VPS4A mutant defected in hexamerization (mVPS4A), but not VPS4B, were also found to interact with CHMP4C and ANCHR. However, VPS4A-ANCHR interactions were abolished in VTA1 KO cells, suggesting the formation of a VPS4A-VTA1-ANCHR complex at the abscission checkpoint. Collectively, our data indicate that VPS4 A and B isoforms have distinct functions remodeling the ESCRT-III filament during mediated cytokinetic abscission that could be partially compensated by the presence of one isoform. However, the regulatory role of VPS4 in abscission is mediated by the VPS4A isoform, potentially in its monomeric form, and is dependent on VTA1 binding. Notably, the regulatory role of VPS4A shown here provide for the first time a mechanistic explanation for the previously reported direct involvement of VPS4A (but not VPS4B) in disease development [ 6 , 7 ].

Cytokinetic abscission provides an excellent platform for dissecting the role of VPS4 paralogs. During abscission, components of the ESCRT-III complex assemble into helical filaments at the intercellular bridge that connect the 2 dividing daughter cells, and drive constriction and fission of the membranes connecting these cells to complete the division process. VPS4 participates in several crucial aspects of this process. First, it is involved in the exchange of ESCRT-III monomers within the filament during filament assembly [ 16 ]. Second, it plays a role in ESCRT-III depolymerization and membrane fission that terminates abscission [ 17 , 18 ]. Third, it interacts with proteins of the AuroraB abscission checkpoint (ANCHR and CHMP4C), which regulates abscission timing [ 19 , 20 ]. Notably, both paralogs were shown to localize at the intercellular bridge when overexpressed in cells [ 16 , 18 ], suggesting their functional involvement in abscission. Therefore, VPS4 exhibits multiple functions in abscission, with both VPS4 paralogs potentially contributing to these functions.

VPS4 is an AAA-ATPase that assembles into functional hexamers or dodecamers that hydrolyze ATP [ 10 , 11 ]. This functional hexamer is stabilized by binding to its co-factor VTA1, which dimerizes to form a bridge that connects 2 adjacent VPS4 subunits [ 11 , 12 ]. While yeast cells encode for a single VPS4 protein, mammalian cells encode for 2 VPS4 homologs, namely VPS4A and VPS4B. Given the high sequence similarity of these paralogs (81% amino acids identity), they were traditionally thought to have redundant functions in cells [ 8 , 13 ]. However, recent evidence suggests that the cellular functions of VPS4A and VPS4B do not fully overlap. First, mutations in VPS4A in patients carrying normal VPS4B caused severe diseases associated with structural brain abnormalities, neurodevelopmental defects, cataract, growth defects, and congenital dyserythropoietic anemia (CDA) [ 6 , 7 ]. Second, while VPS4A was reported to function as a tumor suppressor, VPS4B exhibited pro- or anti-oncogenic activities in different cancers [ 8 , 9 ]. Nevertheless, depleting VPS4A in cancer cells with loss of VPS4B led to synthetic cell death, suggesting at least partial overlap between these isoforms [ 8 ]. Third, VPSB was previously shown to be more crucial for HIV viral release than VPS4A, and we recently substantiated these findings and showed that depletion of VPS4A or VPS4B has differential effects on HIV-viral release, suggesting a unique property for each isoform [ 14 , 15 ]. Collectively, these reports raise the possibility that the 2 VPS4 isoforms hold both overlapping and unique functions in cells stressing the need to investigate the specific function differences of each isoform in cells.

VPS4 is one of the core components of the endosomal sorting complex required for transport (ESCRT) membrane remodeling system, which is currently recognized as one of the most basic cellular machineries for driving membrane fission in cells. As such, VPS4 is essential for in vitro reconstitution of ESCRT-induced membrane fission and is involved in all canonical ESCRT-mediated processes in eukaryotes, including in multivesicular body (MVB) formation, release of retroviruses from the cell surface, nuclear envelope sealing, plasma membrane repair, neural pruning, and scission of daughter cells during the last stages of cytokinesis [ 1 – 3 ]. Additional non-canonical roles for VPS4 in centrosomes have also been reported [ 4 , 5 ]. Finally, mutations in VPS4 genes were associated with different neuro-pathologies and cancer [ 6 – 9 ]. Therefore, understanding the basis for VPS4 function in cells is of great interest.

Results

To dissect the role of VPS4 isoforms in cytokinesis, we monitored abscission in VPSA and VPS4B KO cells recently generated in our laboratory (S1A Fig) [14]. Attempts to generate cells depleted of both VPS4A and VPS4B resulted in cell death, suggesting that at least 1 VPS4 isoform is needed for cell viability, as previously suggested [8]. Depleting either VPS4A or VPS4B caused a delay in abscission and an increase in the percentage of cells that failed to complete abscission but did not affect the formation or morphology of the intercellular bridge (Fig 1A and 1B). VPS4A KO cells exhibited a considerably more severe abscission delay (averaged abscission time of 154 min in VPS4A KO versus 107 min in VPS4B KO compared to 83 min in WT cells). No increase in multinucleated cells was observed in VPS4A KO cells, suggesting the overall cytokinesis is not significantly affected (S1B Fig). Exogenously expressed VPS4A arrived at the intercellular bridge and fully rescued the abscission delay phenotype (Figs 1C and 1D, and S1C). Expressing exogenous VPS4B at similar levels (S1D Fig), only partially rescued the abscission delay (Fig 1C and 1D), indicating that VPS4B cannot fully compensate for the loss of VPS4A in abscission. Moreover, measurements of the total levels of endogenous VPS4 paralogs in WT cells revealed that VPS4B is considerably more abundant than VPS4A (approximately 5:1, respectively), indicating that depletion of VPS4A has smaller effect on the overall levels of VPS4 levels in the cell compared to VPS4B depletion (S1E Fig). Collectively, these findings suggest that, irrespectively to its expression levels, VPS4A is the more prominent paralog in abscission.

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TIFF original image Download: Fig 1. Knock-out of VPS4A causes a stronger abscission delay phenotype than VPS4B knock-out. (A) Live cell imaging of WT (top panel), VPS4B KO (middle panel), and VPS4A KO (bottom panel), transfected with GFP-tubulin. Z slices were captured at 7-min intervals using a confocal spinning-disk microscope. Maximum-intensity projections of representative cells at various time points during cytokinesis are shown. The abscission time was set as time = 0 and was defined as the time of the first cleavage event of the microtubule bridge. Scale, 10 μm. (B) Duration of abscission (from cleavage furrow formation to microtubule bridge cleavage) as measured for WT VPS4A KO and VPS4B KO cells. Top panel, a cumulative plot; bottom panel, a violin plot showing the distribution of abscission times. Averaged abscission times: VPS4A KO 154 ± 45 min, n = 25; VPS4B KO 107 ± 37 min, n = 30; WT 63 ± 25 min, n = 63. Data for each condition were obtained from at least 3 independent experiments (S1 Data), and p values were calculated using Anova (Kruskal–Wallis test). (C) The abscission delay observed in VPS4A KO cells is fully rescued by exogenous expression of VPS4A and partially rescued by overexpression of VPS4B. VPS4A KO cells were co-transfected with mCherry-tubulin (red) together with either GFP-VPS4A (top panel, green) or GFP-VPS4B (bottom panel, green), and imaged 48 h later, at 7-min intervals. Left panels: zoom-out images (scale, 10 μm). Zoom-in images of the white squares in left panels are shown on the right (scale, 5 μm). Shown are maximum-intensity projection images of representative cells. Note the arrival of VPS4A and VPS4B to the intercellular bridge in VPS4A KO cells. Complete movie series are provided in S1 Movie (top panel) and S2 Movie (bottom panel). (D) Duration of abscission (from cleavage furrow formation to microtubule bridge cleavage) as measured for VPS4A KO cells expressing either VPS4A or VPS4B. Left panel, a cumulative plot. Right panel, a violin plot showing the distribution of abscission times. WT and VPS4A KO cell data (reproduced from Fig 1A and 1B) are shown for reference. Averaged abscission times: VPS4A KO + GFP-VPS4A 85 ± 22 min, n = 17; VPS4A KO + GFP-VPS4B 100 ± 28 min, n = 17. Similar abscission rates were observed in WT cells expressing either GFP-tubulin or mCherry-tubulin (S1C Fig). Data were obtained from 5 independent experiments of each condition (S1 Data), and p values were calculated using Anova (Kruskal–Wallis test). https://doi.org/10.1371/journal.pbio.3002327.g001

During cytokinetic abscission, components of the ESCRT-III complex were shown to assemble into specific high-ordered structures of rings and spiral at the intracellular bridge at different stages of abscission [18,21,22]. Structured illumination microscopy (SIM) imaging of the ESCRT-III proteins IST1 and CHMP4B in VPS4 KO cells revealed that the overall organization of the ESCRT-III filaments at the intercellular bridge is not perturbed upon depletion of either isoform (Figs 2A and S2A). The typical ring organization could be readily detected in early bridges of cells depleted of VPS4A or VPS4B and elongation of the ESCRT-III polymer toward the cell body was observed in late bridges in both KO cells, supporting proper spiral formation. We therefore concluded that 1 VPS4 isoform is sufficient for proper ESCRT-III organization at the intercellular bridge.

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TIFF original image Download: Fig 2. Differential effect of VPS4A and VPS4B depletion on the distribution of ESCRT-III at the intercellular bridge. (A) SIM imaging shows normal ESCRT-III organization at the intercellular bridge in VPS4 KO cells. WT (top panel), VPS4A KO (middle panel), and VPS4B KO (bottom panel) cells were stained with anti-α-tubulin (green) and anti-IST1 (red) antibodies and imaged by SIM. Similar results were obtained using anti-CHMP4B (S2A Fig). Shown are maximum projection images of representative cells obtaining early (left) and late (right) intercellular bridges. Data was reproduced in at least 3 independent experiments. Scale, 1 μm. (B) STORM analysis shows different densities of the ESCRT-III protein IST1 at intercellular bridges of VPS4A and VPS4B KO cells. Cells were synchronized, stained with anti-α-tubulin and anti-IST1 antibodies, and imaged by 2D STORM in epifluorescence mode (see Methods). Top panel: wide field images showing the intercellular bridge and IST1 staining (tubulin, green; IST1, white); middle panel: the STORM datasets of IST1 localizations obtained for bridges in top panel; bottom panel: a scatter plot graph showing the density of IST1 at the intercellular of all cells measured. IST1 density was calculated by dividing the total number of localizations by the area of manually selected regions (ROIs) that include IST1 staining on one side of the bridge (red marking, middle left panel). Plots showing area and number of localizations measured for each ROI at the different conditions are presented in S2B Fig. Calculations were performed on data from 5 independent experimental repeats, and p values were calculated using unpaired t test. WT: early, n = 26, late, n = 23; VPSA KO: early, n = 22, late, n = 33; VPSB KO: early, n = 20, late, n = 22 (S2 Data). n refers to the number of ROIs. ESCRT, endosomal sorting complex required for transport; SIM, structured illumination microscopy; STORM, stochastic optical reconstruction microscopy. https://doi.org/10.1371/journal.pbio.3002327.g002

As VPS4 was previously shown to mediate the exchange of proteins within the ESCRT-III polymer, we next set to examine the distribution of ESCRT-III proteins at the intercellular bridge in WT and KO cells [16,23]. To this end, we established a STORM-based assay that allowed to quantify the distribution of endogenous ESCRT-III proteins at the intercellular bridge at the single molecule level (see Methods section). Measurements of the number of localization and the total area that contain IST1 signal at the intercellular bridge revealed an increase in the transition from early to late bridges, in both WT and KO cells, supporting the proper ESCRT-III organization obtained by SIM (S2B Fig). Yet, calculations of the density of IST1 at the intercellular bridge pointed to a differential role of VPS4 paralogs in abscission. In WT cells, a decrease in IST1 density was observed in the transition from early to late intercellular bridges, indicating removal of IST1 proteins from the ESCRT-III structure (Fig 2B). While this phenomenon was recapitulated in VPS4A KO cells, it was almost completely abolished in VPS4B KO cells, suggesting that the decrease in IST1 density in late intercellular bridges is driven by the VPS4B isoform. Comparison of the initial IST1 density in early intercellular bridges of WT and KO cells showed that in the absence of VPS4A, but not of VPS4B, IST density at early intercellular bridges is significantly increased, pointing to the involvement of VPS4A in early stages of abscission (Fig 2B). Collectively, these results suggest that both VPS4 isoforms are involved in IST1 dynamics in the ESCRT-III structure. However, while the VPS4A isoform is primarily involved in early stages the VPS4B isoform act in late stages. Compromising either of these properties does not appear to affect the overall organization of the ESCRT-III polymer but may contribute to the delayed abscission phenotype observed in these KO cells. Therefore, VPS4A and VPS4B appear to have temporally separated functions in abscission.

Previous studies indicated a role for VPS4 in the AuroraB abscission checkpoint [19,20,24]. We therefore asked which of the VPS4 isoforms is involved in this checkpoint. In IP experiments, VPS4A, but not VPS4B, bound the abscission checkpoint proteins ANCHR and CHMP4C (Fig 3A and 3B), indicating that the regulatory role, previously reported for VPS4 in abscission, is mediated by VPS4A. In cells, VPS4 resides in a monomeric or dimeric forms at the cytosol and assemble into active hexamers upon binding to membrane-bound ESCRT-III [25]. To examine whether the regulatory role of VPS4A in abscission relies on hexamerization, we generated a VPS4A monomeric mutant based on a previously reported yeast monomeric VPS4 mutant (mVPS4A) (L145D, corresponds to L151D in yeast VPS4, see S3A Fig) [26]. We found, that similar to WT VPS4A, the mVPS4A mutant bind the AuroraB checkpoint proteins ANCHR and CHMP4C (Fig 3A). Moreover, a fluorescently tagged mVPS4A mutant localized at the intercellular bridge in both WT and VPS4A KO cells, indicating that VPS4A can be targeted to the intracellular bridge in its monomeric form (Fig 3C). Expression of mVPS4A in VPS4A KO cells also resulted a slight increase in abscission rate and a complete recovery in the percentages of cells that completed abscission (72% in VPS4A KO versus 91% in mVPS4A OE and 94% in WT cells) (Figs 3D and S3B). These data indicate that the regulatory role of VPS4 in abscission is facilitated by the VPS4A isoform and may be executed by its monomeric form. It, therefore, appears that VPS4A has a dual function in abscission: one that is regulatory and can be mediated by its monomeric form and another that is associated with its canonical function as an AAA-ATPase that remodels the ESCRT-III filament.

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TIFF original image Download: Fig 3. VPS4A is the paralog involved in the abscission checkpoint. (A, B) WT cells co-transfected with either GFP-VPS4A, GFP-mVPS4A (A), or GFP-VPS4B (B) together with HA-ANCHR (top panels) or mCherry-CHMP4C (bottom panels) were subjected to IP using anti-GFP beads. Note that VPS4A and mVPS4A mutant (VPS4A L145D) bound the abscission checkpoint proteins, whereas VPS4B does not. Immunoblotting: IB fractions - anti-HA (top panels) or anti-mCherry (bottom panels); IP fractions - anti-GFP. Data were reproduced in at least 2 independent experiments. Complete blots are provided in S1 Raw Images. (C) mVPS4A (VPA4A L145D) localizes to the intracellular bridge in WT (top panel) and VPS4A KO (bottom panel) cells. Shown are intercellular bridges of cells that were co-transfected with mCherry-tubulin (red) and GFP-mVPS4A (green). Left panels: zoom-out (scale, 10 μm) images. Zoom-in images of the white squares in left panels are shown on the right (scale, 5 μm). Shown are maximum-intensity projection images of representative cells. Data was reproduced in at least 3 independent experiments. (D) Kinetics of abscission in VPS4A KO cells expressing the mVPS4A mutant. Left panel: maximum-intensity projection time-lapse images of dividing VPS4A KO cells co-transfected with mCherry-tubulin and GFP-mVPS4A (scale, 10 μm). The abscission time, which was defined as the time of the first microtubule bridge cleavage event, was set as time 0. Right panel: a cumulative plot of abscission duration (from cleavage furrow formation to microtubule bridge cleavage) of VPS4A KO expressing mVPS4A (averaged time 138 ± 33 min, S3B Fig). Data was obtained from 2 independent experiments, n = 23 cells (S1 Data). WT and VPS4A KO cell data (reproduced from Fig 1A and 1B) are shown for reference. IP, immunoprecipitation. https://doi.org/10.1371/journal.pbio.3002327.g003

VTA1 is a co-factor of VPS4, which was shown to stabilize its active, hexameric form [11,12,27–29]. To test whether the regulatory role of VPS4 can be induced by destabilizing the VPS4 hexamer, we examined the effect of VTA1 depletion on VPS4 function in dividing cells using a VTA1 KO cell line [14] (S4A Fig). In native polyacrylamide gel electrophoresis, lower molecular weight bands were obtained for VPS4 in VTA1 KO cells compared to WT cells, supporting decreased levels of hexameric VPS4 in VTA1 KO cells (S4B Fig). Unexpectedly, loss of VTA1 did not result in a delay in abscission but instead led to accelerated abscission (Fig 4A). This phenotype was not associated a significant increase in multinucleated cells (S1B Fig). Exogenously expressed VTA1 arrived at the intercellular bridge in VTA1 KO cells and reduced the rate of abscission (Fig 4A and 4B). The cellular levels of VPS4A and VPS4B were not significantly affected in these cells and both proteins properly localized at the intercellular bridge, indicating that VTA1 is dispensable for targeting VPS4 to the intercellular bridge (S4C and S4D Fig). Additionally, no changes in the organization and levels of IST1 at the intercellular bridge were observed, suggesting that the ESCRT-III complex is not significantly affected (S4E and S4F Fig). Therefore, despite the canonical role of VTA1 in stabilizing the active VPS4 hexamer, its depletion did not significantly affect the arrival and organization of ESCRT-III-VPS4 proteins to the intercellular bridge or lead to delayed abscission.

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TIFF original image Download: Fig 4. VTA1 is part of the abscission checkpoint complex. (A) Abscission is faster in cells depleted of VTA1. Live cell imaging of VTA1 KO cells transfected with GFP-tubulin alone or with mCherry-tubulin and mEmerald-VTA1. Shown are maximum-intensity projections of tubulin labeling from time-lapse movies of representative cells. Time 0 = abscission time. Colored images on bottom panel demonstrate the arrival of VTA1 to the intercellular bridge during abscission in VTA1 KO cells (see also S3 Movie). Scale, 5 μm. (B) Duration of abscission (from cleavage furrow formation to microtubule bridge cleavage) as measured for VTA1 KO and VTA1 KO cells expressing exogenous VTA1 are shown of right panels. Top panel, a cumulative plot; bottom panel, a violin plot showing the distribution of abscission times. Averaged duration times of abscission were: VTA1 KO 73.3 ± 30 min, n = 32; VTA1 KO + VTA1 102 ± 39 min, n = 23 (S1 Data). WT data (reproduced from Fig 1A and 1B) is shown for reference. Data were obtained from at least 3 independent experiments, and p values were calculated using Anova (Kruskal–Wallis test). (C) Accumulation of chromatin bridges in VTA1 KO cells. Representative images of VTA1 KO cells stained with anti-α-tubulin (green) and Hoechst (blue) are shown. Left panel: DNA staining. Arrows indicate chromatin bridges. Second panel: a Laplacian 2D filter analysis applied to the DNA staining images for better visualization of DNA bridges. Third panel: A merged image of tubulin and DNA staining. Scale, 10 μm. Right panel: A plot showing the percentages of intercellular bridges cells with DNA bridges in WT, VTA1 KO, and VTA1 KO cells expressing mEmerald-VTA1 (WT; n = 654, VTA1 KO; n = 454, VTA1 KO + VTA1; n = 784 cells) (S1 Data). Data was reproduced in at least 2 independent experiments, and p values were calculated using chi square. (D) VTA1 binds the abscission checkpoint proteins CHMP4C and ANCHR. WT cells co-transfected with mEmerald-VTA1 and HA-ANCHR (left panel) or mCherry-CHMP4C (right panel) were subjected to IP using anti-GFP beads. Immunoblotting: IB fractions anti-HA (left panels) or anti-mCherry (right panels), IP fractions anti-GFP antibodies. (E) WT cells co-transfected with the indicated GFP-tagged VPS4 constructs, and mApple-VTA1 were subjected to IP using anti-GFP beads. Immunoblotting: IB fractions anti-VTA1 (arrow indicates the VTA1 band), IP fractions anti-GFP. Note that VTA1 preferentially binds to VPS4A in cells. Data in C and D were reproduced in at least 2 independent experiments. Complete blots of panels D and E are provided in S1 Raw Images. IP, immunoprecipitation. https://doi.org/10.1371/journal.pbio.3002327.g004

Accelerated abscission was previously reported upon depletion of abscission checkpoint proteins, suggesting the involvement of VTA1 in the abscission checkpoint [1,24]. An increase in chromatin bridges, which results from chromosome misseggregation, was previously associated with failure of the abscission checkpoint and specifically with disfunction of the CHMP4C and ANCHR [20,24]. Consistent with a role for VTA1 in the abscission checkpoint, the percentage of intercellular bridges that contained lagging chromatin was significantly increased in VTA1 KO cells, and this phenotype was restored upon exogenous VTA1 expression (Fig 4C). Levels of the abscission checkpoint protein AuroraB were similar in WT and VTA1 KO cells, suggesting that VTA1 does not affect the arrival of AuroraB to the intercellular bridge (S4G Fig). In IP experiments performed in WT cells, VTA1 was found to interact with the checkpoint proteins ANCHR and CHMP4C (Fig 4D). VTA1 could also bind VPS4B and VPS4A, with stronger binding affinity observed for the latter, and was associated with the mVPS4A (Fig 4E). To further investigate the involvement of VTA1 in the VPS4-CHMP4C-ANCHR abscission checkpoint complex, we analyzed the interactions between VPS4, VTA1, ANCHR, and CHMP4C in the KO cell lines (Fig 5A–5C). Interestingly, we found that the interaction between VPS4A and ANCHR was lost in VTA1 KO cells, while its binding to CHMP4C was unperturbed (Fig 5A and 5C). On the other hand, in VPS4A KO cells, VTA1 lost its ability to interact with CHMP4C but maintained binding to ANCHR (Fig 5B and 5C). Collectively, these findings strongly suggest that VTA1 is an integral part of a CHMP4C/ANCHR/VPS4A abscission checkpoint complex and is involved in the regulation of abscission timing.

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