Synaptopodin stress fiber and contractomere at the epithelial junction

Abstract

The apical junction of epithelial cells can generate force to control cell geometry and perform contractile processes while maintaining barrier function and adhesion. Yet, the structural basis for force generation at the apical junction is not fully understood. Here, we describe two synaptopodin-dependent actomyosin structures that are spatially, temporally, and structurally distinct. The first structure is formed by the retrograde flow of synaptopodin initiated at the apical junction, creating a sarcomeric stress fiber that lies parallel to the apical junction. Contraction of the apical stress fiber is associated with either clustering of membrane components or shortening of junctional length. Upon junction maturation, apical stress fibers are disassembled. In mature epithelial monolayer, a motorized “contractomere” capable of “walking the junction” is formed at the junctional vertex. Actomyosin activities at the contractomere produce a compressive force evident by actin filament buckling and measurement with a new α-actinin-4 force sensor. The motility of contractomeres can adjust junctional length and change cell packing geometry during cell extrusion and intercellular movement. We propose a model of epithelial homeostasis that utilizes contractomere motility to support junction rearrangement while preserving the permeability barrier.

Synaptopodin is necessary forShigella flexneriintercellular spread

“…T84 cells were grown in Advance DMEM supplemented with 10% FBS and 1% L-glutamine. The generation of T84 cells producing control or synaptopodin specific shRNA were previously described (11), and the generation of MDCK cells producing control or synaptopodin specific shRNA were previously described (12). HeLa cells producing shRNA targeting synaptopodin were generated using retroviral transduction followed by selection with 5 µg/mL puromycin.…”

“…Whereas the number of plaques is a function of the efficiency of bacterial invasion, the area of the plaques is a function of the efficiency of intercellular spread. We measured the area of plaques formed in T84 human colonic adenocarcinoma epithelial (T84) cells and Madin-Darby canine kidney (MDCK) cells that either produced a control shRNA or a shRNA specific to synaptopodin (11,12). Synaptopodin protein levels are reduced by 80% in the T84 cells (11) and 85% in MDCK cells (Fig.…”

“…We further sought to examine whether we could identify interactions that were important for the formation of the protrusion. Synaptopodin is described to interact with myosin X and a-actinin-4 at the plasma membrane, where together they form the contractomere complex that generates force so as to enable the extrusion of cells from a monolayer (12). Myosin X is required for the formation of protrusions by S. flexneri (29), and recent work shows that a-actinin-4 is present at actin structures formed during S.…”

“…It promotes barrier integrity of the intestinal epithelium in response to butyrate by localizing to tight junctions, where it interacts with actin fibers (11). In MDCK cells, synaptopodin promotes cellular extrusion by forming a complex known as a contractomere, which comprises synaptopodin, a-actinin-4, b-catenin, and myosin IIb and generates contractile forces along the cell junction (12). Due to the critical role of actin-binding proteins during infection by cytosolic pathogens, we sought to test whether and how synaptopodin contributes to infection.…”

Modulation of Piezo1 influences human skin architecture and oxytocin expression

“…E‐cadherins participate in mechano‐sensation, information transduction, allowing tissues to remodel themselves in response to a change in the environment [9]. The apical part of keratinocytes also participates in mechanical sensation, with the involvement of synaptopodin fibres and of α‐actinin‐4 as a force sensor [10]. The nucleus plays a key role in mechanotransduction, environmental forces could be transmitted to the nucleus via the “linker of nucleoskeleton and cytoskeleton” complex (LINC) affecting the nuclear shape and density of heterochromatin [11].…”