Dissection of the mammalian midbody proteome reveals conserved cytokinesis mechanisms

Abstract

Cytokinesis is the essential process that partitions cellular contents into daughter cells. To identify and characterize cytokinesis proteins rapidly, we used a functional proteomic and comparative genomic strategy. Midbodies were isolated from mammalian cells, proteins were identified by multidimensional protein identification technology (MudPIT), and protein function was assessed in Caenorhabditis elegans. Of 172 homologs disrupted by RNA interference, 58% displayed defects in cleavage furrow formation or completion, or germline cytokinesis. Functional dissection of the midbody demonstrated the importance of lipid rafts and vesicle trafficking pathways in cytokinesis, and the utilization of common membrane cytoskeletal components in diverse morphogenetic events in the cleavage furrow, the germline, and neurons.

Fig. 1

Isolation of CHO cell midbodies. (A) Synchronized CHO cells in the late stages of cytokinesis. (B) Isolated midbodies following biochemical preparation. In addition to bipolar midbodies (left, top inset), multipolar spindle midbodies (bottom inset) were also obtained, likely resulting from polyploid CHO cells. (C) Silver-stained gel showing the complex protein composition of isolated midbodies separated by SDS–polyacrylamide gel electrophoresis. (D) Western blot showing enrichment of candidate proteins in midbodies (MID) isolated from synchronized cells as compared to a fraction prepared from unsynchronized interphase cells (INT) treated in the same manner. Each lane contained 10 μg of total protein and was probed with antibodies recognizing Dyn2 (dynamin II), α-tubulin, KHC (kinesin heavy chain), or BiP.

Fig. 2

Distribution of functional categories of the identified proteins and RNAi phenotypes of the C. elegans homologs. (A) Pie chart displaying the categories of mammalian proteins identified by midbody mass spectrometry analysis (total number of proteins = 577). Candidate midbody proteins (160) chosen for further study are in red. Mitochondrial, ribosomal, nuclear, transcription/translation, and heat shock proteins also identified in the midbody preparations were not pursued in this study (table S2). (B) Pie chart illustrating the percentage of mammalian proteins in each functional category. (C) Bar graph displaying the percentage of C. elegans genes targeted by RNAi in each phenotype category. Some genes gave multiple phenotypes and were therefore listed in multiple categories. (D) The percentage of genes with cell division phenotypes belonging to specific functional classes.

Fig. 3

Localization of putative midbody proteins to the midbody in mammalian cells. Immunofluorescence showing 10 candidate midbody proteins previously uncharacterized with respect to the midbody. GM130 marks the localization of the Golgi in the midbody.

Fig. 4

Examples of cytokinesis and germline cytokinesis defects following RNAi of midbody components. (A to D) Wild-type (WT) embryo; (E to H) K04D7.1-RACK1 RNAi. (A and E) Pronuclei meet in the center of the embryo. (B and F) Spindle sets up on the anterior-posterior axis [cytoplasmic clearing is apparent in (F)]. (C and G) Cleavage furrow ingresses. (D) WT embryo continued to divide (Movie S1). (H) K04D7.1-RACK1–inhibited embryo failed to complete cytokinesis and the furrow regressed (Movie S2). Scale bar, 5 μm. (I to P) Germline cytokinesis defects resulting from RNAi of midbody components. (I) Top focal plane, and (M) mid-focal plane of WT gonad showing organized nuclei at the periphery and a central region devoid of nuclei (the rachis) in the distal portion of the gonad. (J) Enlargement of top focal plane and (N) mid-focal plane of a WT gonad. (K) Top focal plane of W02G9.2-Keap1 gonad, showing germ cells with multiple nuclei. (O) Mid-focal plane of W02G9.2-Keap1, showing a diminished rachis and multinucleate germ cells that appear larger than normal. (L) Mid-focal plane of T01G1.3-SEC31 gonad, showing nuclei within the rachis. (P) Mid-focal plane of F09C3.1-IQGAP gonad, showing multinucleate cellularized germ cells. Scale bar, 5 μm.

Fig. 5

Examples of meiotic and chromosome segregation defects after RNAi treatment. (A) WT embryo with two polar bodies (arrowheads) extruded. (B) W02G9.2-Keap1–deficient embryo in which one polar body failed to be extruded, resulting in three nuclei (marked with asterisks) within the embryo. An arrowhead marks the extruded polar body. (C) F09C3.1-IQGAP–depleted embryo in which both polar bodies failed to extrude (four nuclei marked with asterisks). (D to F) Montage of metaphase through anaphase in a WT embryo expressing histone H2B::GFP (Movie S3). (D) A normal metaphase plate. (E and F) Anaphase. (G to I) Montage of T05E11.3-Endoplasmin–depleted embryo expressing histone H2B::GFP that displayed chromosome segregation defects (Movie S4). Note the lack of chromosome congression to the metaphase plate in (G) and lagging chromosomes in (H) and (I) during anaphase (marked by arrowheads). Scale bar, 5 μm.