Dysregulation of the endoplasmic reticulum blocks recruitment of centrosome-associated proteins resulting in mitotic failure.

The endoplasmic reticulum (ER) undergoes a remarkable transition in morphology during cell division to aid in the proper portioning of the ER. However, whether changes in ER behaviors modulate mitotic events is less clear. Like many animal embryos, the early Drosophila embryo undergoes rapid cleavage cycles in a lipid-rich environment. Here, we show that mitotic spindle formation, centrosomal maturation, and ER condensation occur with similar time frames in the early syncytium. In a screen for Rab family GTPases that display dynamic function at these stages, we identified Rab1. Rab1 disruption led to an enhanced buildup of ER at the spindle poles and produced an intriguing 'mini-spindle' phenotype. ER accumulation around the mitotic space negatively correlates with spindle length/intensity. Importantly, centrosomal maturation is defective in these embryos, as mitotic recruitment of key centrosomal proteins is weakened after Rab1 disruption. Finally, division failures and ER overaccumulation is rescued by Dynein inhibition, demonstrating that Dynein is essential for ER spindle recruitment. These results reveal that ER levels must be carefully tuned during mitotic processes to ensure proper assembly of the division machinery.

A Rab39-Klp98A-Rab35 endocytic recycling pathway is essential for rapid Golgi-dependent furrow ingression.

Ingression of the plasma membrane is an essential part of the cell topology-distorting repertoire and a key element in animal cell cytokinesis. Many embryos have rapid cleavage stages in which they are furrowing powerhouses, quickly forming and disassembling cleavage furrows on timescales of just minutes. Previous work has shown that cytoskeletal proteins and membrane trafficking coordinate to drive furrow ingression, but where these membrane stores are derived from and how they are directed to furrowing processes has been less clear. Here, we identify an extensive Rab35/Rab4>Rab39/Klp98A>trans-Golgi network (TGN) endocytic recycling pathway necessary for fast furrow ingression in the Drosophila embryo. Rab39 is present in vesiculotubular compartments at the TGN where it receives endocytically derived cargo through a Rab35/Rab4-dependent pathway. A Kinesin-3 family member, Klp98A, drives the movements and tubulation activities of Rab39, and disruption of this Rab39-Klp98A-Rab35 pathway causes deep furrow ingression defects and genomic instability. These data suggest that an endocytic recycling pathway rapidly remobilizes membrane cargo from the cell surface and directs it to the trans-Golgi network to permit the initiation of new cycles of cleavage furrow formation.

Sponge/DOCK-dependent regulation of F-actin networks directing cortical cap behaviors and syncytial furrow ingression.

In the early syncytial Drosophila embryo, rapid changes in filamentous actin networks and membrane trafficking pathways drive the formation and remodeling of cortical and furrow morphologies. Interestingly, genomic integrity and the completion of mitoses during cell cycles 10-13 depends on the formation of transient membrane furrows that serve to separate and anchor individual spindles during division. While substantial work has led to a better understanding of the core network components that are responsible for the formation of these furrows, less is known about the regulation that controls cytoskeletal and trafficking function. The DOCK protein Sponge was one of the first proteins identified as being required for syncytial furrow formation, and disruption of Sponge deeply compromises F-actin populations in the early embryo, but how this occurs is less clear. Here, we perform quantitative analysis of the effects of Sponge disruption on cortical cap growth, furrow formation, membrane trafficking, and cytoskeletal network regulation through live-imaging of the syncytial embryo. We find that membrane trafficking is relatively unaffected by the defects in branched actin networks that occur after Sponge disruption, but that Sponge acts as a master regulator of a diverse cohort of Arp2/3 regulatory proteins. As DOCK family proteins have been implicated in regulating GTP exchange on small GTPases, we also suggest that Rac GTPase activity bridges Sponge regulation to the regulators of Arp2/3 function. Finally, we demonstrate the phasic requirements for branched F-actin and linear F-actin networks in potentiating furrow ingression. In total, these results provide quantitative insights into how a large DOCK scaffolding protein coordinates the activity of a variety of different actin regulatory proteins to direct the remodeling of the apical cortex into cytokinetic-like furrows.