Unravelling the spindle-independent function of cortical force-generating machinery (LIN-5/GPR-1/2) during cleavage furrow formation and abscission in C. elegans embryos

Abstract

Cytokinesis marks the final stage of cell division, where a single mother cell divides into two daughter cells. This intricate process begins with the assembly of an actomyosin-based cleavage furrow at the equatorial membrane during anaphase and culminates in abscission, which ensures the proper distribution of cellular contents into the newly formed daughter cells. In animal cells, the appropriate position of the cleavage furrow strictly relies on the spatiotemporal accumulation of a small GTPase RhoA at the equatorial membrane. RhoA directly regulates actin dynamics and indirectly controls myosin II activity, thus helping in cleavage furrow assembly. The RhoA-based contractile ring invariably assembles and ingresses in the middle of the mitotic spindle. Protein complexes localized at the spindle midzone positively regulate the accumulation of RhoA at the equatorial membrane. Notably, in many cells, including C. elegans embryos, the spindle midzone is present at a significant distance from the equatorial membrane. Thus, it has been unclear how the assembly and the timing of contractile ring formation are regulated in such cells. In this context, the cortically anchored machinery comprises G/GPR-1/2/LIN-5 (G/LGN/NuMA in humans), which is critical for cortical pulling force generation and, thus, for proper spindle positioning, is implicated in cleavage furrow initiation. However, because of the relevance of these proteins in a myriad of distinct processes, including aster separation, spindle positioning, and myosin II removal from the membrane, it has been elusive if these proteins dictate cleavage furrow formation directly, or indirectly via their spindle-dependent activity. In this study, we used the C. elegans embryos as a model system to investigate the role of the evolutionarily conserved protein LIN-5 (NuMA in humans) in cytokinesis. In the first part of my thesis, I focused on testing the spindle-independent role of LIN-5 (or GPR-1/2) in promoting cleavage furrow formation. In the second part, I discovered the novel role of the LIN-5-based complex in maintaining the stability of the midbody and, thus, abscission.

1. Cortical accumulation of LIN-5-based complexes control cleavage furrow formation In this part, I uncovered that the cortical accumulation of LIN-5/GPR-1/2 (referred to as LIN-5-based complexes) at the polar region of the cell membrane regulates furrow formation independent of its role in 1) aster separation, 2) spindle positioning, and 3) its recently proposed function in myosin II removal from the polar cortical region. Instead, our data suggest that the enriched levels of LIN-5/GPR-1/2 at the polar cortical surface control the equatorial accumulation of myosin II. Overall, this part of my thesis offers a mechanistic understanding of how LIN-5/GPR-1/2 at the polar cortical membrane establishes a focused furrow-forming zone at the right time during cell division.

2. A novel role of cortical LIN-5-based complexes in facilitating midbody stability and, therefore, abscission As the cleavage furrow ingresses, the spindle midzone undergoes remodeling to form a densely packed midbody. This region accumulates many proteins essential for making the final cut for the abscission. One quintessential midbody component is an evolutionary conserved, heterotetrameric protein called centralspindlin complex composed of ZEN-4 (MKLP1 in humans) and CYK-4 (MgcRacGAP in humans). Centralspindlin plays a role in cleavage furrow formation as well as in midbody stability. Abscission fails in cells where the centralspindlin cannot accumulate at the midbody post-furrow ingression. Interestingly, we found LIN-5-based complexes robustly accumulate at the membrane post-furrow ingression in the vicinity of the midbody and the midbody surrounding membrane. We show that the cortical accumulation of LIN-5-based complexes in concert with the spindle midzone ensures the stability of the midbody. In embryos that are compromised for the spindle midzone, LIN-5 or GPR-1/2 depletion leads to loss of centralspindlin complex from the midbody followed by abscission failure. Overall, these results indicate that cortical accumulation of LIN-5/GPR-1/2 complexes helps to confine the contractile ring components to the midbody surrounding membrane, thereby securing midbody stabilization for error-free abscission.