Actin dynamics drive microvillar motility and clustering during brush border assembly

Abstract

During differentiation, transporting epithelial cells generate large arrays of microvilli known as a brush borders to enhance functional capacity. To develop our understanding of brush border formation, we used live cell imaging to visualize apical surface remodeling during early stages of this process. Strikingly, we found that individual microvilli exhibit persistent active motility, translocating across the cell surface at ~0.2 μm/min. Perturbation studies with inhibitors and photokinetic experiments revealed that microvillar motility is driven by actin assembly at the barbed-ends of core bundles, which in turn is linked to robust treadmilling of these structures. Because the apical surface of differentiating epithelial cells is crowded with nascent microvilli, persistent motility promotes collisions between protrusions and ultimately leads to their clustering and consolidation into higher order arrays. Thus, microvillar motility represents a previously unrecognized driving force for apical surface remodeling and maturation during epithelial differentiation.3

Nonmuscle myosin-2 contractility-dependent actin turnover limits the length of epithelial microvilli

“…Using a combination of chemical and genetic perturbations to increase or decrease the activity of this motor, we observed striking shortening or elongation of microvilli, respectively ( Figure 6 ). Interestingly, previous studies from our laboratory revealed a similar effect of NM2 inhibition (with blebbistatin) on the nascent microvilli that exhibit robust translocation across the apical surface of LLC-PK1-CL4 kidney epithelial cells early in differentiation ( Meenderink et al. , 2019 ).…”

“…Time-lapse analysis of W4 cells treated with 20 µM blebbistatin revealed that microvilli, which normally exhibit striking dynamics on the apical surface (e.g., elongation, shortening, and waving or pivoting around the base), immediately become static and subsequently begin to elongate (Supplemental Movie 3). A previous study from our group established that the parallel actin bundles that support microvilli exhibit robust treadmilling (i.e., retrograde flow), where actin monomer incorporation at tip-oriented barbed ends is balanced by subunit removal from the pointed ends that emerge from the base of these structures ( Meenderink et al. , 2019 ).…”

“…, 2019 ). Early in differentiation, this treadmilling activity is coupled to directed motion of microvilli across the apical surface, which in turn promotes the adherent packing and organization of these protrusions ( Meenderink et al. , 2019 ).…”

“…, 2019 ). In that context, inhibition of NM2 did not impair forward movement of microvilli, which was found to be driven by actin incorporation into core bundle filaments, although it did lead to the elongation of motile protrusions from their trailing ends ( Meenderink et al. , 2019 ).…”

“…How does NM2C activity promote the shortening of microvillar actin bundles? Previous work established that growing and nascent microvilli are supported by highly dynamic core bundles of parallel actin filaments, which collectively exhibit robust treadmilling ( Tyska and Mooseker, 2002 ; Meenderink et al. , 2019 ).…”