Capping protein regulates actin dynamics during cytokinetic midbody maturation

Abstract

During cytokinesis, a cleavage furrow generated by actomyosin ring contraction is restructured into the midbody, a platform for the assembly of the abscission machinery that controls the final separation of daughter cells. The polymerization state of F-actin is important during assembly, ingression, disassembly, and closure of the contractile ring and for the cytoskeletal remodeling that accompanies midbody formation and progression to abscission. Actin filaments must be cleared from the abscission sites before the final cut can take place. Although many conserved proteins interact with and influence the polymerization state of actin filaments, it is poorly understood how they regulate cytokinesis in higher eukaryotes. We report here that the actin capping protein (CP), a barbed end actin binding protein, participates in the control of actin polymerization during later stages of cytokinesis in human cells. Cells depleted of CP furrow and form early midbodies, but they fail cytokinesis. Appropriate recruitment of the ESCRT-III abscission machinery to the midbody is impaired, preventing the cell from progressing to the abscission stage. To generate actin filaments of optimal length, different actin nucleators, such as formins, balance CP's activity. Loss of actin capping activity leads to excessive accumulation of formin-based linear actin filaments. Depletion of the formin FHOD1 results in partial rescue of CP-induced cytokinesis failure, suggesting that it can antagonize CP activity during midbody maturation. Our work suggests that the actin cytoskeleton is remodeled in a stepwise manner during cytokinesis, with different regulators at different stages required for successful progression to abscission.

EphB2 and ERK signaling are required for heterotypic contact inhibition of locomotion to drive cell sorting

“…This is in stark contrast to fibrosarcoma cells (HT1080) which, upon collision with epithelial cells, seemed to undergo a complete repulsion (Figure 1A, B and Video S1). This result was not only seen with HaCaT epithelial cells, as fibrosarcoma collision with a human corneal epithelial cell line [5] also resulted in repulsion of the fibrosarcoma population (Figure S1). When no colliding partner was encountered, HaCaT epithelial cells migrated with a persistently increasing speed, which was similarly observed after their collision with fibrosarcoma cells, suggesting that epithelial cell motion was unaffected by their collision with the fibrosarcoma population (Figure 1C, D).…”

“…Immortalized human keratinocytes (HaCaT), immortalized mouse fibroblasts (NIH3T3), human fibrosarcoma (HT1080) and immortalized human corneal epithelial cells [5] were all maintained in Dulbecco’s Modified Eagle’s Medium (DMEM) with 4500 mg / L glucose, supplemented with 10 % FBS, at 37° C and 5 % CO 2 . For routine maintenance, cells were cultured in T75 plastic cell culture flasks and split via trypsinization approximately every 3 days or when approaching confluence.…”

Human AKTIP interacts with ESCRT proteins and functions at the midbody in cytokinesis

“…We then asked whether the AKTIP localization was temporally and spatially linked to subunits of the ESCRT machinery. We focused on the elements of the ESCRT III complex IST1, CHMP4B and CHMP2A, for which the spatial and temporal localization at the midbody had been previously defined (7, 10, 20). In early forming midbodies, a low AKTIP signal follows the tubulin profile (Fig.…”

RNAi Transfection Results in Lipidome Changes

“…Therefore, HeLa cells were treated with the TRs alone or complexed with non‐targeting (NT) siRNA for 72 h (Figure a,b). To confirm that the experimental conditions result in successful RNAi, we optimized our transfection protocols using target‐specific siRNAs for three cell division‐associated proteins we routinely study in our lab (RACGAP1, Anillin, and CAPZB, Figure S1, Supporting Information) . Immunoblot analyses of total cell lysates illustrate that our protocol achieves penetrant protein knock down and that the knock down efficiency is comparable for both TRs (Figure S1, Supporting Information).…”