The Golgi-associated protein GRASP65 regulates spindle dynamics and is essential for cell division.
At the onset of mitosis, the pericentriolar Golgi apparatus of mammalian cells is converted into small fragments, which are dispersed throughout the cytosol. The Golgi-associated protein GRASP65 is involved in this process. To address the role of GRASP65 in mitotic Golgi fragmentation, we depleted the protein from HeLa cells by RNAi. In the absence of GRASP65, the number of cisternae per Golgi stack is reduced without affecting the overall organization of Golgi membranes and protein transport. GRASP65-depleted cells entered mitosis, but accumulated in metaphase with condensed chromatin and multiple aberrant spindles and eventually died. Although Centrin2 and g-tubulin were detected in two of the spindle poles, the other spindle poles contained g-tubulin, but not Centrin2. Furthermore, we provide evidence that the expression of the C-terminus of GRASP65 interferes with entry of cells into mitosis. Our results suggest the requirement for GRASP65 in the regulation of spindle dynamics rather than a direct role in the stacking of Golgi cisternae. This novel function is in addition to the previously established negative role of GRASP65 at the G2/M transition, which is mediated by its C-terminus.
Sütterlin
Christine
C
Cell and Developmental Biology, University of California-San Diego, La Jolla, CA 92093-0347, USA.
Polishchuk
Roman
R
Pecot
Matt
M
Malhotra
Vivek
V
D001483:Base Sequence; D001692:Biological Transport; D002453:Cell Cycle; D002455:Cell Division; D045744:Cell Line, Tumor; D002469:Cell Separation; D002843:Chromatin / Q000378:metabolism; D003600:Cytosol / Q000378:metabolism; D005434:Flow Cytometry; D016195:G2 Phase; D006056:Golgi Apparatus / Q000378:metabolism*; D000077614:Golgi Matrix Proteins; D006367:HeLa Cells; D006801:Humans; D047468:Immunoprecipitation; D008562:Membrane Glycoproteins / Q000378:metabolism; D008565:Membrane Proteins / Q000378:metabolism / Q000502:physiology*; D008677:Metaphase; D008854:Microscopy, Electron; D008856:Microscopy, Fluorescence; D016253:Microscopy, Immunoelectron; D008938:Mitosis; D008969:Molecular Sequence Data; D015739:Nocodazole / Q000494:pharmacology; D017434:Protein Structure, Tertiary; D021381:Protein Transport; D034622:RNA Interference; D008941:Spindle Apparatus; D013997:Time Factors; D014162:Transfection; D014759:Viral Envelope Proteins / Q000378:metabolism
D002843:Chromatin; C028391:G protein, vesicular stomatitis virus; C108705:GORASP1 protein, human; D000077614:Golgi Matrix Proteins; D008562:Membrane Glycoproteins; D008565:Membrane Proteins; D014759:Viral Envelope Proteins; D015739:Nocodazole