William M. Brieher

Actin networks in cells are dynamic and constantly turning over as actin subunits exchange between monomer and polymer pools. Understanding these dynamics in vivo requires a detailed understanding of pure actin behavior in vitro. The prevailing model is treadmilling, which predicts continuous growth of filament barbed ends balanced by continuous shrinkage of pointed ends, while filament length remains roughly constant. While treadmilling has been observed directly at the level of single filaments, length fluctuations—expressed as “length diffusivity”—are much greater than expected, and the origin of these high diffusivity values could not be identified experimentally. By imaging single filaments tethered to glass via α-actinin, we observed frequent, low-amplitude barbed-end fluctuations of ±2–6 subunits per event and rarer, high-amplitude fluctuations of ±50–150 subunits per event. Barbed-end fluctuations depended on adenosine triphosphate (ATP) hydrolysis and release of inorganic phosphate (Pi) and were blocked by phalloidin and barbed-end capping agents. Barbed-end fluctuations consumed an estimated one-third of the ATP at steady state, while two-thirds is still consumed by treadmilling. Fluctuations result in diffusive spreading of filament lengths that exceeds predictions from pure treadmilling yet is lower than previously reported values, suggesting that earlier measurements captured additional sources of variability. Our results provide direct experimental evidence for kinetic models that proposed a dampened form of dynamic instability in pure actin where transient loss of the ATP/ADP•Pi barbed-end cap leads to rare, large excursions superimposed on more frequent, small fluctuations.

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.

Significance: A major question in tissue biology is how cells manage to maintain stable cell–cell adhesion using the intrinsically weak homophilic bonds of cadherin adhesion molecules. One suggestion is that cells cluster cadherins into large puncta to promote strong adhesion. However, we now show that puncta are not clusters of cadherins but rather interdigitated protruding microspikes. Actin polymerization–dependent protrusive activity generates the interdigitated fingers that help hold the cells together by increasing the membrane surface area available for cadherin–cadherin molecular interactions and by generating a rough, interlocking topography to impede relative movement between cells. The latter, analogous to friction, may offer a physical means of adhesion to compliment the chemical adhesion afforded by cadherin–cadherin homophilic binding.

The apical junction of epithelial cells can generate force to control cell geometry and perform contractile processes while maintaining barrier function and cell-cell adhesion. Yet, the structural basis of force generation at the apical junction is not completely understood. Here, we describe 2 actomyosin structures at the apical junction containing synaptopodin, myosin IIB, and alpha-actinin-4. We showed that synaptopodin is required for the assembly of E-cadherin-associated apical stress fibers and a novel macromolecular structure, which we named contractomere. Knockdown of synaptopodin abolished both apical stress fiber and contractomere formation. Moreover, depletion of synaptopodin abolished basal stress fibers, converting myosin IIA sarcomere-like arrangement into a meshwork-type actomyosin organization. We propose a new model of junction dynamics that is dependent on contractomere movement to control epithelial cell boundary and geometry. Our findings reveal 2 actomyosin structures at the epithelial junction and underscore synaptopodin in the assembly of stress fibers and contractomeres. Summary Statement Synaptopodin assembles 2 actomyosin structures at the epithelial junction: apical stress fiber and contractomere. Synaptopodin selectively regulates myosin IIB without altering the level of myosin IIA and is responsible for converting evolutionary-conserved actomyosin meshwork into vertebrate-specific stress fibers.

Cadherins build stable, cohesive sheets of cells using paradoxically weak bonds. Actin is thought to convert weak binding into strong adhesion either by transmitting myosin dependent pulling forces to adhesive junctions or by clustering cadherins in the plane of the membrane. Here, however, we show that continuous actin polymerization stabilizes cadherin adhesion by directly driving membrane protrusions, not by promoting contractility or cadherin clustering. Lateral membranes of epithelial cells are continuously pushed against each another by protrusions. Micrometer sized cadherin puncta, long thought to be clusters of cadherins, turn out to be patches of microspikes interlocked by cadherin homophilic bonds to hold neighboring cells together. When actin polymerization is blocked, protrusions cease, puncta disappear, and lateral membranes detach from one another. In contrast, inhibiting myosin II contractility has no effect on adhesion. One Sentence Summary: Stronger together: membrane interdigitations keep cells attached.

Significance: All solid tissues rely on cadherin family cell–cell adhesion molecules for their cohesion and organization. Cadherin adhesive function depends on the actin cytoskeleton, but actin’s contribution to cell–cell adhesion is not fully understood. We demonstrate that actin polymerization-dependent protrusive activity operates continuously to push lateral membranes of neighboring cells together to keep cadherins in contact. Actin-dependent protrusive activity functions as a safety mechanism to quickly repair cadherin adhesive junctions whenever they fail and to prevent myosin-dependent contractile forces from tearing the junctions further apart. Since loss of cadherin adhesion is associated with a number of diseases including cancer progression, then misregulation of actin protrusive activity should be considered as a possible contributing factor to epithelial pathophysiology.

Maintaining the correct ratio of apical, basal, and lateral membrane domains is important for epithelial physiology. Here, we show that CD2AP is a critical determinant of epithelial membrane proportions. Depletion of CD2AP or phosphoinositide 3-kinase (PI3K) inhibition results in loss of F-actin and expansion of apical–basal domains, which comes at the expense of lateral membrane height in MDCK cells. We demonstrate that the SH3 domains of CD2AP bind to PI3K and are necessary for PI3K activity along lateral membranes and constraining cell area. Tethering the SH3 domains of CD2AP or p110γ to the membrane is sufficient to rescue CD2AP-knockdown phenotypes. CD2AP and PI3K are both upstream and downstream of actin polymerization. Since CD2AP binds to both actin filaments and PI3K, CD2AP might bridge actin assembly to PI3K activation to form a positive feedback loop to support lateral membrane extension. Our results provide insight into the squamous to cuboidal to columnar epithelial transitions seen in complex epithelial tissues in vivo.

Cadherin mediated cell-cell adhesion is actin dependent, but the precise role of actin in maintaining cell-cell adhesion is not fully understood. Actin polymerization-dependent protrusive activity is required to push distally separated cells close enough together to initiate contact. Whether protrusive activity is required to maintain adhesion in confluent sheets of epithelial cells is not known. By electron microscopy as well as live cell imaging, we have identified a population of protruding actin microspikes that operate continuously near apical junctions of polarized MDCK cells. Live imaging shows that microspikes containing E-cadherin extend into gaps between E-cadherin clusters on neighboring cells while reformation of cadherin clusters across the cell-cell boundary triggers microspike withdrawal. We identify Arp2/3, EVL, and CRMP-1 as three actin assembly factors necessary for microspike formation. Depleting these factors from cells using RNAi results in myosin II-dependent unzipping of cadherin adhesive bonds. Therefore, actin polymerization-dependent protrusive activity operates continuously at cadherin cell-cell junctions to keep them shut and to prevent myosin II-dependent contractility from tearing cadherin adhesive contacts apart.