Myotubularin-related proteins 3 and 4 interact with polo-like kinase 1 and centrosomal protein of 55 kDa to ensure proper abscission

Abstract

The myotubularins are a family of phosphatases that dephosphorylate the phosphatidylinositols phosphatidylinositol-3-phosphate and phosphatidylinositol-3,5-phosphate. Several family members are mutated in disease, yet the biological functions of the majority of myotubularins remain unknown. To gain insight into the roles of the individual enzymes, we have used affinity purification coupled to mass spectrometry to identify protein-protein interactions for the myotubularins. The myotubularin interactome comprises 66 high confidence (false discovery rate ≤1%) interactions, including 18 pairwise interactions between individual myotubularins. The results reveal a number of potential signaling contexts for this family of enzymes, including an intriguing, novel role for myotubularin-related protein 3 and myotubularin-related protein 4 in the regulation of abscission, the final step of mitosis in which the membrane bridge remaining between two daughter cells is cleaved. Both depletion and overexpression of either myotubularin-related protein 3 or myotubularin-related protein 4 result in abnormal midbody morphology and cytokinesis failure. Interestingly, myotubularin-related protein 3 and myotubularin-related protein 4 do not exert their effects through lipid regulation at the midbody, but regulate abscission during early mitosis, by interacting with the mitotic kinase polo-like kinase 1, and with centrosomal protein of 55 kDa (CEP55), an important regulator of abscission. Structure-function analysis reveals that, consistent with known intramyotubularin interactions, myotubularin-related protein 3 and myotubularin-related protein 4 interact through their respective coiled coil domains. The interaction between myotubularin-related protein 3 and polo-like kinase 1 relies on the divergent, nonlipid binding Fab1, YOTB, Vac1, and EEA1 domain of myotubularin-related protein 3, and myotubularin-related protein 4 interacts with CEP55 through a short GPPXXXY motif, analogous to endosomal sorting complex required for transport-I components. Disruption of any of these interactions results in abscission failure, by disrupting the proper recruitment of CEP55, and subsequently, of endosomal sorting complex required for transport-I, to the midbody. Our data suggest that myotubularin-related protein 3 and myotubularin-related protein 4 may act as a bridge between CEP55 and polo-like kinase 1, ensuring proper CEP55 phosphorylation and regulating CEP55 recruitment to the midbody. This work provides a novel role for myotubularin-related protein 3/4 heterodimers, and highlights the temporal and spatial complexity of the regulation of cytokinesis.

Fig. 1.

The myotubularin interactome. Stable HEK293 Flp-In T-REx cell lines expressing FLAG-tagged versions of 15 human myotubularin-related proteins were generated, and protein interactions for each were identified by AP-MS. Two biological replicates were performed for each myotubularin, and compared with 18 FLAG-GFP controls. Statistical analysis was performed using SAINTexpress. All interactions shown had a false discovery rate (FDR) ≤1%.

Fig. 2.

MTMR3 and MTMR4 interact with PLK1 and CEP55. A, Stable HEK293 Flp-In T-REx cell lines expressing FLAG-tagged CEP55 or PLK1 were analyzed by AP-MS as in Fig. 1. Information on additional interactions can be found at prohits-web.lunenfeld.ca. B, HEK293T cells were transfected with HA-MTMR3 or HA-MTMR4, along with FLAG-tagged GFP, MTMR3, MTMR4, PLK1, and CEP55. After 24 h, cells were lysed and subjected to FLAG-immunoprecipitation. Western blot analysis using HA and FLAG antibodies were used to detect interactions between components.

Fig. 3.

MTMR3 and MTMR4 are both required for abscission. A, HeLa and U-2 OS cells were treated with siRNA against MTMR3, MTMR4, both myotubularins together, or CEP55. After 72 h, cells were fixed, stained with α-Tubulin antibody and DAPI, and analyzed for binucleation. B, U-2 OS Flp-In T-REx cells stably expressing FLAG-tagged GFP, GFP-MTMR3, or GFP-MTMR4 were induced with 1 μg/ml tetracycline, fixed and stained 72 h later, and analyzed for binucleation. C, HeLa cells were transfected with siRNA targeting each of the 16 myotubularins, and incubated 72 h before fixation and staining. D, Stable U-2 OS cell lines with inducible expression of FLAG-GFP, siRNA-resistant GFP-MTMR3, or siRNA-resistant GFP-MTMR4 were transfected with control, MTMR3, or MTMR4 siRNA. Twenty-four hours later protein expression was induced with 0.01 μg/ml tetracycline (note the decreased tetracycline added to the medium to prevent the overexpression effects observed in Fig. 3B). Cells were fixed and stained 48 h later, and analyzed for abscission defects. E, Fate of mitosis of HeLa cells depleted of CEP55, MTMR3, or MTMR4 was assayed by live cell imaging 48 h after siRNA transfection. The number of mitoses analyzed for each condition (n) is indicated. F, The lengths of the microtubule midbody bridge in cytokinetic HeLa cells depleted of CEP55, MTMR3, or MTMR4 were measured. At least 100 midbodies were measured per condition. G, Postmitotic cells with continued membrane attachment after MTMR3, MTMR4, or CEP55 depletion are shown, stained for α-Tubulin to visualize interphase microtubules, CEP55, and the ESCRT-III cofactor VPS4B. Scale bars: 10 μm (full images), 2 μm (zoomed images). H, U-2 OS Flp-In T-REx cells were treated as in D, and assessed for the presence of postmitotic membrane fusions. Results for all binucleation assays, as well as the membrane fusion assay, are the average of three biological replicates, with least 300 cells were analyzed per treatment for each replicate.

Fig. 4.

Proper abscission does not require MTMR3/MTMR4 lipid phosphatase activity, but does require interactions between MTMR3, MTMR4, PLK1, and CEP55. A, U-2 OS Flp-In T-REx cell lines with inducible expression of GFP-tagged CEP55, MTMR3, or MTMR4 were fixed and immunostained with GFP and α-Tubulin antibodies. Scale bars: 10 μm (full images), 2 μm (zoomed images). B, Schematic of MTMR3 functional domains. The black X in the MTMR3 FYVE domain denotes its inability to bind phospholipids. U-2 OS Flp-In T-REx cell lines with inducible expression of siRNA-resistant, GFP-tagged MTMR3 were transfected with either control or MTMR3 siRNA and incubated 24 h before protein expression was induced with 0.01 μg/ml tetracycline. 72 h after RNA transfection, cells were fixed and stained for α-Tubulin and GFP, and analyzed for abscission defects. C, Schematic of MTMR4 functional domains. U-2 OS Flp-In T-REx cell lines with inducible expression of siRNA-resistant, GFP-tagged MTMR4 lines were analyzed as in panel B. For B and C, three biological replicates were performed, and at least 300 cells were counted per sample in each replicate. D, U-2 OS Flp-In T-REx cell lines with inducible expression of siRNA-resistant, GFP-tagged MTMR3 or MTMR4 point mutations were subjected to Western blot analysis. For MTMR3, mutations are as follows: PHmut, L73P; PTPmut, C413S; CCmut, V1049/L1052/L1056P; FYVEmut, C1174S. For MTMR4, GPPYmut, Y11A; PHmut, L73P; PTPmut, C407S; CCmut, V1036/L1039/V1043/L1046P; FYVEmut, C1169S. PH, PH-GRAM; PTP, protein tyrosine phosphatase; CC, coiled coil; FYVE, FYVE domain; GPPY, GPPXXXY motif. E, HEK293T cells were transfected with FLAG-tagged wild type or coiled coil mutant versions of MTMR3 or MTMR4, and lysed after 24 h. Because of the lack of MTMR4 antibodies, HA-MTMR4 was cotransfected in the MTMR3 experiment. FLAG immunoprecipitation was performed, and interactions with indicated components were analyzed by Western blot. F, HEK293T cells were transfected with FLAG-tagged wild-type MTMR3, MTMR3-FYVEmut, wild-type MTMR4, or MTMR4-GPPYmut, and lysed after 24 h. FLAG immunoprecipitation was performed, and interactions with PLK1 and CEP55 were analyzed by Western blot. kDa, kilodalton. G, Sequence alignment of ALIX, TSG101, and MTMR4 GPPY motifs. MTMR3 does not contain an analogous sequence. Flanking numbers indicate the amino acid positions of the motif in each protein.

Fig. 5.

MTMR3/MTMR4 heterodimers associate with PLK1 and CEP55 during early mitosis. A, HEK293 Flp-In T-REx cells were transfected with either control or MTMR3 siRNA for 48 h before induction of FLAG-MTMR4 expression for 24 h. Lysates were subjected to FLAG immunoprecipitation and immunoblotted for complex components. B, HEK293 Flp-In T-REx cells were transfected with either control or MTMR4 siRNA for 48 h before induction of FLAG-MTMR4 expression for 24 h. Lysates were subjected to FLAG immunoprecipitation and immunoblotted for complex components. MTMR4 depletion was confirmed by RT-PCR (denoted by *). C, HEK293T cells were transfected with FLAG-MTMR4, and synchronized in various stages of the cell cycle. Lysates were FLAG-immunoprecipitated, and MTMR3, PLK1, and CEP55 interactions were analyzed by Western blot. Cell cycle progression was monitored by immunoblotting the input lysates for phosphorylated serine 10 of Histone H3 (P-H3 S10). Aph, aphidicolin; Noc, nocodazole; kDa, kilodalton. D, HeLa Flp-In T-REx cells with stable expression of either FLAG-MTMR3 or FLAG-MTMR4 were arrested in either S phase or mitosis. Mitotic lysates were then incubated with or without λ phosphatase before Western blot analysis. E, Stable HeLa Flp-In T-REx cell lines were arrested as in D, and mitotic cells were incubated with either 10 μm RO-3066 or 1 μm BI2536 for 30 min before lysis and Western blot analysis.

Fig. 6.

MTMR3 and MTMR4 regulate CEP55 recruitment to the midbody. A, HeLa cells depleted of CEP55, MTMR3, or MTMR4 for 72 h were fixed and immunostained with antibodies against CEP55, α-Tubulin, and PLK1. Midbodies were imaged quantitatively and the total CEP55 intensity at the midbody was measured. B, Fixed images of CEP55 recruitment to the central spindle/midbody through late mitosis after depletion of CEP55, MTMR3, or MTMR4. Scale bars: 10 μm. C, D, HeLa cells were treated as in B, but stained with antibodies against the ESCRT-I component ALIX or the Centralspindalin component MKLP1. E. Stable U-2 OS cell lines with inducible expression of FLAG-GFP or siRNA-resistant forms of GFP-MTMR3 and GFP-MTMR4 were transfected with the indicated siRNAs and incubated 24 h before protein expression was induced with 0.01 μg/ml tetracycline. 48 h after induction, cells were fixed and stained for α-Tubulin, GFP, and CEP55. Midbodies were imaged quantitatively, and total CEP55 intensity at the midbody was measured. For all quantitative imaging experiments, results shown depict one of three independent replicates, with at least 60 midbodies measured per condition per replicate. The red line indicates the mean, the pink box indicates the 95% confidence interval, and the gray box indicates one standard deviation.