Abstract
The mitotic spindle is required for chromosome congression and subsequent equal segregation of sister chromatids. These processes involve a complex network of signaling molecules located at the spindle. The endocytic protein, clathrin, has a "moonlighting" role during mitosis, whereby it stabilizes the mitotic spindle. The signaling pathways that clathrin participates in to achieve mitotic spindle stability are unknown. Here, we assessed the mitotic spindle proteome and phosphoproteome in clathrin-depleted cells using quantitative MS/MS (data are available via ProteomeXchange with identifier PXD001603). We report a spindle proteome that consists of 3046 proteins and a spindle phosphoproteome consisting of 5157 phosphosites in 1641 phosphoproteins. Of these, 2908 (95.4%) proteins and 1636 (99.7%) phosphoproteins are known or predicted spindle-associated proteins. Clathrin-depletion from spindles resulted in dysregulation of 121 proteins and perturbed signaling to 47 phosphosites. The majority of these proteins increased in mitotic spindle abundance and six of these were validated by immunofluorescence microscopy. Functional pathway analysis confirmed the reported role of clathrin in mitotic spindle stabilization for chromosome alignment and highlighted possible new mechanisms of clathrin action. The data also revealed a novel second mitotic role for clathrin in bipolar spindle formation.
Highlights
During mitosis, a mitotic spindle is established to pull sister chromatids toward opposite poles of the dividing cell
Functional pathway analysis of the proteins disrupted by clathrin depletion confirmed the reported role of clathrin in stabilizing the mitotic spindle and revealed a novel second role for clathrin in bipolar spindle formation
We report a comprehensive coverage of the spindle proteome and spindle phosphoproteome
Summary
SiRNA Transfection and Mitotic Spindle Isolation—HeLa S3 cells were grown in RPMI media (Invitrogen, Mulgrave, Australia) containing 10% fetal bovine serum (FBS) (Invitrogen) to a density of 7.5 e6 cells per ml for each treatment (total of three treatments in one experiment). The protein abundance of clathrin and TACC3 is high in HeLa cells, they may have been present at a very low abundance at the spindle compared with whole cell lysates These were not found in the actual files (proteinGroups.txt) used for processing. The average of all peptides found for clathrin from the separately processed phosphopeptide and nonphosphopeptide data sets found in the respective Evidence.txt files were extracted. Phosphopeptides for the protein TACC3 were eliminated from further analysis using the PhosphoSTY files because a) not all peptides passed the internal algorithm criteria and b) those that did pass were found in Ͻ3 biological replicates. Instances that displayed p values Ͻ 0.05 following a t test were considered to be a significant change
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