The Clathrin-dependent Spindle Proteome

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 clathrindepleted 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. Molecular & Cellular

Visualization of human karyopherin beta-1/importin beta-1 interactions with protein partners in mitotic cells by co-immunoprecipitation and proximity ligation assays

“…Many identified interactors localize to the spindle, the main site of importin beta-1 localization in mitosis. Clathrin, a component of the endocytic pathway with RNA-binding capacity, and a recognized example of a moonlighting protein, localizes to MTs and regulates spindle functions, in part by localizing other proteins to the spindle 51 . Interestingly, clathrin targets the MT-regulatory factor TACC-3 to the spindle MTs 52 in an importin-beta-1-sensitive manner: importin beta-1 inhibits clathrin/TACC3 binding, while RANGTP restores it.…”

Proteomic Profiling of Microtubule Self-organization in M-phase

“…This is probably because of intrinsic advantages of the egg extract system that provides a good source of relatively abundant and clean preparations of RanGTP induced MT assemblies. Moreover they can be purified without extensive and long manipulation steps, such as those required for mitotic spindle preparations (8,10). Interestingly, the RanMT proteome also includes 431 proteins that are absent in any of the reported spindle proteomes (8 -11) and 74 of these proteins are spindle proteins included in MiCroKITS.…”

Tau‐based fluorescent protein fusions to visualize microtubules

“…An important consideration when assessing the potential utility of a new cytoskeletal probe is that it does not perturb normal cytoskeletal function. With a long and ever expanding list of MAPs (Bonner et al, 2011;Rao et al, 2016;Sauer et al, 2005) and a limited number of binding sites along the MT lattice, it is somewhat surprising that the addition of exogenous MAPs seems to have minimal effect on the assembly of MT-based structures. Interestingly, we found that spindle size and spindle shape were largely unperturbed in the presence of Tau-based FP fusions (Figure 3e,f).…”