A microtubule interactome: complexes with roles in cell cycle and mitosis

Abstract

The microtubule (MT) cytoskeleton is required for many aspects of cell function, including the transport of intracellular materials, the maintenance of cell polarity, and the regulation of mitosis. These functions are coordinated by MT-associated proteins (MAPs), which work in concert with each other, binding MTs and altering their properties. We have used a MT cosedimentation assay, combined with 1D and 2D PAGE and mass spectrometry, to identify over 250 MAPs from early Drosophila embryos. We have taken two complementary approaches to analyse the cellular function of novel MAPs isolated using this approach. First, we have carried out an RNA interference (RNAi) screen, identifying 21 previously uncharacterised genes involved in MT organisation. Second, we have undertaken a bioinformatics analysis based on binary protein interaction data to produce putative interaction networks of MAPs. By combining both approaches, we have identified and validated MAP complexes with potentially important roles in cell cycle regulation and mitosis. This study therefore demonstrates that biologically relevant data can be harvested using such a multidisciplinary approach, and identifies new MAPs, many of which appear to be important in cell division.

Figure 1. The MT Cosedimentation Assay, and Validation of MAPs

(A) A representation of the methodology behind the assay (see text for details). Only MTs and their associated proteins pellet through the sucrose cushion; all other proteins remain in the supernatant. (B) 2D gel electrophoresis of the MT pellet. Approximately 880 features are visible in the 2D gel. Tubulin migrates as distinct features (e.g., arrow), which were avoided when choosing spots for mass spectrometry. In addition, we avoided cutting more than one feature from sets that clustered together in the same region of the gel (e.g., red box). (C) 1D gel electrophoresis of the MT pellet. Many distinct bands of varying molecular weight are seen. (D and E) Validation of the MAP hits. Antibodies were obtained to a selection of previously characterised proteins and tested for their ability to bind MTs in a cosedimentation assay (D) and to localise to MTs in S2 cells (E). Scale bar indicates 10 μm.

Figure 2. Functional Classification of 270 Drosophila Embryonic MAPs

A pie chart showing the classification of identified MAPs according to Gene Ontologies, assisted by manual data mining of references in Flybase. A total of 31% of proteins were completely uncharacterised according to these sources. The remaining proteins were grouped by primary GO function and were then encompassed into four main categories: MT related (i), DNA/RNA associated (ii), protein folding/degradation (iii), and other (iv).

Figure 3. RNAi Phenotypes Associated with Previously Uncharacterised MAPs

S2 cells were fixed with cold methanol and immunostained for α-tubulin (green), phospho-Histone H3 (unpublished data), and DNA (red), 5 d after being treated with dsRNA corresponding to each gene. A representative image of the phenotype observed in each class of gene identified in Table 1 is shown. (A, C, E, and G) mitotic cells. (B, D, F, H, and I) interphase cells. Dotted lines represent outlines of interphase cells defined by phase contrast observation. Scale bar indicates 10 μm.

Figure 4. Pairwise Interactions of Identified MAPs

(A) A total of 66 of the 270 MAPS show direct pairwise interactions with another MAP, gathered from GRID and Flybase; displayed here as an interaction network. Proteins are indicated by circular nodes and interactions by lines, or edges. (B) The distribution of interactions within randomly selected sets of proteins. The average number of interactions is 42.71 (s.d. ± 13.09; 1,000 repeats). The MAP set shows 92 interactions, 3.77 standard deviations away from the mean of the distribution. (C) A bar chart representing the size and compositions of complexes in (A). Any complex containing either characterised mitotic/cell cycle proteins, or uncharacterised proteins showing an RNAi phenotype are represented here. The largest putative complex (indicated by an asterisk [*]) contains both classes of proteins.

Figure 5. SkpA and SkAP Are Centrosomal MAPs That Form a Complex In Vivo, and Regulate Centrosome Number

(A) An embryonic MT cosedimentation assay probed with antibodies to SkpA and SkAP. A fraction of both proteins are present in the MT pellet in the presence of Taxol. α-Tubulin is shown as a control. (B) Immobilised anti-SkpA antibodies were used to precipitate SkpA from 0–4-h embryo extracts. SkAP, but not a control protein, Pnut, coprecipitates with SkpA. C, control precipitate; P, bound precipitate; T, total embryo extract; U, unbound supernatant. (C) Localisation of SkpA and SkAP in meiotic spermatocytes. Both SkpA and SkAP are present at centrosomes throughout the cell cycle. DNA is shown in blue, and either SkpA or SkAP is shown in red. SkpA is additionally present on the nuclear envelope and on the central spindle MTs during anaphase and telophase. In addition to centrosomes, SkAP is found on mitochondria, which aggregate around the central spindle during telophase (arrow). (D) An S2 cell in metaphase, expressing GFP-SkAP. The fusion protein accumulates at centrosomes. Merged; DNA (blue), MTs (red), GFP-SkAP (green). (E) S2 cells treated with dsRNA against skpA or skap/CG11963. Cells were fixed with methanol and stained to visualise MTs (green), DNA (blue), and centrosomes (red). RNAi against either gene results in supernumerary centrosomes. (F) A bar chart representing the percentage of mitotic cells showing an increase in centrosome number (i.e., more than two centrosomes per cell; dark grey), and cells showing defects in cytokinesis (i.e., binucleate cells; light grey) when treated with control dsRNA, or dsRNA against skpA or skap/CG11963. No increase in binucleate cells is seen, demonstrating that the increase in centrosome number is not due to failure of cytokinesis. Scale bar indicates 10 μm.