Giansanti Maria Grazia, Vanderleest Timothy E, Jewett Cayla E, Sechi Stefano, Frappaolo Anna, Fabian Lacramioara, Robinett Carmen C, Brill Julie A, Loerke Dinah, Fuller Margaret T, Blankenship J Todd
Istituto di Biologia e Patologia Molecolari del CNR, Dipartimento di Biologia e Biotecnologie, Università Sapienza di Roma, Roma, Italy.
Department of Physics, University of Denver, Denver, Colorado, United States of America.
PLoS Genet. 2015 Nov 3;11(11):e1005632. doi: 10.1371/journal.pgen.1005632. eCollection 2015 Nov.
Mitotic and cytokinetic processes harness cell machinery to drive chromosomal segregation and the physical separation of dividing cells. Here, we investigate the functional requirements for exocyst complex function during cell division in vivo, and demonstrate a common mechanism that directs anaphase cell elongation and cleavage furrow progression during cell division. We show that onion rings (onr) and funnel cakes (fun) encode the Drosophila homologs of the Exo84 and Sec8 exocyst subunits, respectively. In onr and fun mutant cells, contractile ring proteins are recruited to the equatorial region of dividing spermatocytes. However, cytokinesis is disrupted early in furrow ingression, leading to cytokinesis failure. We use high temporal and spatial resolution confocal imaging with automated computational analysis to quantitatively compare wild-type versus onr and fun mutant cells. These results demonstrate that anaphase cell elongation is grossly disrupted in cells that are compromised in exocyst complex function. Additionally, we observe that the increase in cell surface area in wild type peaks a few minutes into cytokinesis, and that onr and fun mutant cells have a greatly reduced rate of surface area growth specifically during cell division. Analysis by transmission electron microscopy reveals a massive build-up of cytoplasmic astral membrane and loss of normal Golgi architecture in onr and fun spermatocytes, suggesting that exocyst complex is required for proper vesicular trafficking through these compartments. Moreover, recruitment of the small GTPase Rab11 and the PITP Giotto to the cleavage site depends on wild-type function of the exocyst subunits Exo84 and Sec8. Finally, we show that the exocyst subunit Sec5 coimmunoprecipitates with Rab11. Our results are consistent with the exocyst complex mediating an essential, coordinated increase in cell surface area that potentiates anaphase cell elongation and cleavage furrow ingression.
有丝分裂和胞质分裂过程利用细胞机制来驱动染色体分离和分裂细胞的物理分离。在这里，我们研究了胞外分泌复合体功能在体内细胞分裂过程中的功能需求，并证明了一种共同机制，该机制指导细胞分裂过程中的后期细胞伸长和分裂沟进展。我们表明，洋葱圈（onr）和漏斗蛋糕（fun）分别编码果蝇中Exo84和Sec8胞外分泌亚基的同源物。在onr和fun突变细胞中，收缩环蛋白被招募到分裂精母细胞的赤道区域。然而，胞质分裂在沟侵入早期就被破坏，导致胞质分裂失败。我们使用具有自动计算分析的高时空分辨率共聚焦成像来定量比较野生型与onr和fun突变细胞。这些结果表明，在胞外分泌复合体功能受损的细胞中，后期细胞伸长严重受损。此外，我们观察到野生型细胞表面积的增加在胞质分裂开始几分钟后达到峰值，并且onr和fun突变细胞在细胞分裂期间特别是表面积生长速率大大降低。透射电子显微镜分析显示，onr和fun精母细胞中细胞质星体膜大量积累，正常高尔基体结构丧失，这表明胞外分泌复合体是通过这些区室进行适当囊泡运输所必需的。此外，小GTP酶Rab11和PITP乔托向分裂位点的募集取决于胞外分泌亚基Exo84和Sec8的野生型功能。最后，我们表明胞外分泌亚基Sec5与Rab11共免疫沉淀。我们的结果与胞外分泌复合体介导细胞表面积的必要协调增加一致，这种增加增强了后期细胞伸长和分裂沟侵入。
