CP250, a Novel Acidic Coiled Coil Protein of theDictyosteliumcentrosome, Affects Growth, Chemotaxis, and the Nuclear Envelope

Abstract

The Dictyostelium centrosome is a nucleus associated body consisting of a box-shaped core surrounded by the corona, an amorphous matrix functionally equivalent to the pericentriolar material of animal centrosomes which is responsible for the nucleation and anchoring of microtubules. Here we describe CP250 a component of the corona, an acidic coiled coil protein that is present at the centrosome throughout interphase while disappearing during prophase and reappearing at the end of late telophase. Amino acids 756-1148 of the 2110 amino acids are sufficient for centrosomal targeting and cell cycle-dependent centrosome association. Mutant cells lacking CP250 are smaller in size, growth on bacteria is delayed, chemotaxis is altered, and development is affected, which, in general, are defects observed in cytoskeletal mutants. Furthermore, loss of CP250 affected the nuclear envelope and led to reduced amounts and altered distribution of Sun-1, a conserved nuclear envelope protein that connects the centrosome to chromatin.

Functional characterization of CP148, a novel key component for centrosome integrity in Dictyostelium

“…Elucidation of the molecular processes underlying these substantial morphological events requires information about the proteins involved. Several corona components such as centrin, c-tubulin complex proteins (ctubulin, Spc97, Spc98), the XMAP215-family protein CP224, TACC, EB1, LIS1, and CP248/250 have been thoroughly investigated [10][11][12][13][14][15][16][17]. We were recently able to identify four novel components of the central core structure through a proteomic screen (CP39, CP55, CP75, and CP91) [15,18].…”

The Dictyostelium discoideum GPHR Ortholog Is an Endoplasmic Reticulum and Golgi Protein with Roles during Development

“…GPHR-GFP also colocalized with the Golgi apparatus marker comitin, detected by MAb 190-340 (24). Comitin strongly stained the Golgi membranes in the vicinity of the nucleus and overlapped with the microtubule organizing center, from which microtubules labeled by MAb YL1/2 (19) originated and which was stained with monoclonal antibodies recognizing centrosomal protein CP250 (31). The PDI-specific antibodies did not label the GPHR-GFP-decorated membranes associated with Golgi membranes (Fig.…”

“…N-and O-glycosylation of csA was detected with MAb 123-353, which primarily recognizes N-glycosylated csA, and MAb 24-210, detecting O-glycosylated proteins (29), with MAb 188-19 directed against cap32 (30) as a loading control. The centrosome was labeled with MAb K68-332-3, detecting CP250 (31). In Western blots, proteins were detected with enhanced chemiluminescence using horseradish peroxidase-coupled secondary antibodies.…”

Redundant and unique roles of coronin proteins in Dictyostelium

“…Analysis was with a Leica TCS SP5 confocal microscope (Leica, Wetzlar, Germany). For analysis of mitotic microtubule structures, cells were treated with nocodazole (10 lM) for 3 h [27]. Cells were fixed with cold methanol, microtubules were stained with the a-tubulin antibody YL1/2 [28], nuclei were stained with DAPI.…”