Abstract
Cell division is essential for the maintenance of life and involves chromosome segregation and subsequent cytokinesis. The processes are tightly regulated at both the spatial and temporal level by various genes, and failures in this regulation are associated with oncogenesis. Here, we investigated the gene responsible for defects in cell division by using murine temperature-sensitive (ts) mutant strains, tsFT101 and tsFT50 cells. The ts mutants normally grow in a low temperature environment (32 °C) but fail to divide in a high temperature environment (39 °C). Exome sequencing and over-expression analyses identified Diaph3, a member of the formin family, as the cause of the temperature sensitivity observed in tsFT101 and tsFT50 cells. Interestingly, Diaph3 knockout cells showed abnormality in cytokinesis at 39 °C, and the phenotype was rescued by re-expression of Diaph3 WT, but not Diaph1 and Diaph2, other members of the formin family. Furthermore, Diaph3 knockout cells cultured at 39 °C showed a significant increase in the level of acetylated α-tubulin, an index of stabilized microtubules, and the level was reduced by Diaph3 expression. These results suggest that Diaph3 is required for cytokinesis only under high temperature conditions. Therefore, our study provides a new insight into the mechanisms by which regulatory factors of cell division function in a temperature-dependent manner.
Highlights
Cytokinesis occurs after partitioning the duplicated chromosomes into two nuclei, and these nuclei were separated into daughter cells by the cleavage furrow
We focused on diaphanous related formin 3 (Diaph3) among the genes, since the exome analysis revealed that the Diaph3 mutation in tsFT101 cells is the missense homozygous mutation in which I733 is replaced by asparagine (DIAPH3I733N) on the formin homology 2 (FH2) domain, which is the actin nucleating domain (Figure S1) [13]
The growth curve analysis indicated that KO-Diaph1 and KO-Diaph2 cells did not fully recover, unlike KO-Diaph3 cells (Figure 4F,G). These results suggest that Diaph3, but not other formins, restores the temperature sensitivity of Diaph3 KO cells
Summary
Cytokinesis occurs after partitioning the duplicated chromosomes into two nuclei, and these nuclei were separated into daughter cells by the cleavage furrow (review articles [1,2]). The complexes activate downstream signaling proteins including the key guanine nucleotide exchange factor Rho-GEF named Ect-2, Rho family GTPases, and proteins that act locally to assemble a contractile ring, which is assembled around the equator of the dividing cells. The contractile ring was composed of actin filaments and myosin-II and assembled as shown below. The primary signal to assemble the contractile ring comes from Ect-2 that activates the Rho-GTPase. The active Rho-GTPase drives the contractile ring assembly in animal cells by activating Rho-kinase (ROCK) to phosphorylate the regulatory light chains of myosin-II. The active Rho-GTPase drives the assemble of actin filaments into the contractile ring by activating formins, which have instrumental roles in controlling rearrangements of the actin cytoskeleton. Interactions of myosin-II with actin filaments produce force to assemble and constrict the contractile ring to form the cleavage furrow. Contractile rings disassemble as they constrict, resulting in the completion of cytokinesis
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