Mitotic spindle positioning protein (MISP) preferentially binds to aged F-actin.

Actin bundling proteins crosslink filaments into polarized structures that shape and support membrane protrusions including filopodia, microvilli, and stereocilia. In the case of epithelial microvilli, mitotic spindle positioning protein (MISP) is an actin bundler that localizes specifically to the basal rootlets, where the pointed ends of core bundle filaments converge. Previous studies established that MISP is prevented from binding more distal segments of the core bundle by competition with other actin-binding proteins. Yet whether MISP holds a preference for binding directly to rootlet actin remains an open question. By immunostaining native intestinal tissue sections, we found that microvillar rootlets are decorated with the severing protein, cofilin, suggesting high levels of ADP-actin in these structures. Using total internal reflection fluorescence microscopy assays, we also found that purified MISP exhibits a binding preference for ADP- versus ADP-Pi-actin-containing filaments. Consistent with this, assays with actively growing actin filaments revealed that MISP binds at or near their pointed ends. Moreover, although substrate attached MISP assembles filament bundles in parallel and antiparallel configurations, in solution MISP assembles parallel bundles consisting of multiple filaments exhibiting uniform polarity. These discoveries highlight nucleotide state sensing as a mechanism for sorting actin bundlers along filaments and driving their accumulation near filament ends. Such localized binding might drive parallel bundle formation and/or locally modulate bundle mechanical properties in microvilli and related protrusions.

Molecular counting of myosin force generators in growing filopodia.

Animal cells build actin-based surface protrusions to enable biological activities ranging from cell motility to mechanosensation to solute uptake. Long-standing models of protrusion growth suggest that actin filament polymerization provides the primary mechanical force for "pushing" the plasma membrane outward at the distal tip. Expanding on these actin-centric models, our recent studies used a chemically inducible system to establish that plasma membrane-bound myosin motors, which are abundant in protrusions and accumulate at the distal tips, can also power robust filopodial growth. How protrusion resident myosins coordinate with actin polymerization to drive elongation remains unclear, in part because the number of force generators and thus, the scale of their mechanical contributions remain undefined. To address this gap, we leveraged the SunTag system to count membrane-bound myosin motors in actively growing filopodia. Using this approach, we found that the number of myosins is log-normally distributed with a mean of 12.0 ± 2.5 motors [GeoMean ± GeoSD] per filopodium. Together with unitary force values and duty ratio estimates derived from biophysical studies for the motor used in these experiments, we calculate that a distal tip population of myosins could generate a time averaged force of ∼tens of pN to elongate filopodia. This range is comparable to the expected force production of actin polymerization in this system, a point that necessitates revision of popular physical models for protrusion growth. SIGNIFICANCE STATEMENT: This study describes the results of in-cell molecular counting experiments to define the number of myosin motors that are mechanically active in growing filopodia. This data should be used to constrain future physical models of the formation of actin-based protrusions.

Protrusion growth driven by myosin-generated force.

Actin-based protrusions extend from the surface of all eukaryotic cells, where they support diverse activities essential for life. Models of protrusion growth hypothesize that actin filament assembly exerts force for pushing the plasma membrane outward. However, membrane-associated myosin motors are also abundant in protrusions, although their potential for contributing, growth-promoting force remains unexplored. Using an inducible system that docks myosin motor domains to membrane-binding modules with temporal control, we found that application of myosin-generated force to the membrane is sufficient for driving robust protrusion elongation in human, mouse, and pig cell culture models. Protrusion growth scaled with motor accumulation, required barbed-end-directed force, and was independent of cargo delivery or recruitment of canonical elongation factors. Application of growth-promoting force was also supported by structurally distinct myosin motors and membrane-binding modules. Thus, myosin-generated force can drive protrusion growth, and this mechanism is likely active in diverse biological contexts.

An early-life microbiota metabolite protects against obesity by regulating intestinal lipid metabolism.

The mechanisms by which the early-life microbiota protects against environmental factors that promote childhood obesity remain largely unknown. Using a mouse model in which young mice are simultaneously exposed to antibiotics and a high-fat (HF) diet, we show that Lactobacillus species, predominant members of the small intestine (SI) microbiota, regulate intestinal epithelial cells (IECs) to limit diet-induced obesity during early life. A Lactobacillus-derived metabolite, phenyllactic acid (PLA), protects against metabolic dysfunction caused by early-life exposure to antibiotics and a HF diet by increasing the abundance of peroxisome proliferator-activated receptor γ (PPAR-γ) in SI IECs. Therefore, PLA is a microbiota-derived metabolite that activates protective pathways in the small intestinal epithelium to regulate intestinal lipid metabolism and prevent antibiotic-associated obesity during early life.

Translational implications of Th17-skewed inflammation due to genetic deficiency of a cadherin stress sensor.

Desmoglein 1 (Dsg1) is a cadherin restricted to stratified tissues of terrestrial vertebrates, which serve as essential physical and immune barriers. Dsg1 loss-of-function mutations in humans result in skin lesions and multiple allergies, and isolated patient keratinocytes exhibit increased proallergic cytokine expression. However, the mechanism by which genetic deficiency of Dsg1 causes chronic inflammation is unknown. To determine the systemic response to Dsg1 loss, we deleted the 3 tandem Dsg1 genes in mice. Whole transcriptome analysis of embryonic Dsg1-/- skin showed a delay in expression of adhesion/differentiation/keratinization genes at E17.5, a subset of which recovered or increased by E18.5. Comparing epidermal transcriptomes from Dsg1-deficient mice and humans revealed a shared IL-17-skewed inflammatory signature. Although the impaired intercellular adhesion observed in Dsg1-/- mice resembles that resulting from anti-Dsg1 pemphigus foliaceus antibodies, pemphigus skin lesions exhibit a weaker IL-17 signature. Consistent with the clinical importance of these findings, treatment of 2 Dsg1-deficient patients with an IL-12/IL-23 antagonist originally developed for psoriasis resulted in improvement of skin lesions. Thus, beyond impairing the physical barrier, loss of Dsg1 function through gene mutation results in a psoriatic-like inflammatory signature before birth, and treatment with a targeted therapy significantly improved skin lesions in patients.

Loss of myosin Vb promotes apical bulk endocytosis in neonatal enterocytes.

In patients with inactivating mutations in myosin Vb (Myo5B), enterocytes show large inclusions lined by microvilli. The origin of inclusions in small-intestinal enterocytes in microvillus inclusion disease is currently unclear. We postulated that inclusions in Myo5b KO mouse enterocytes form through invagination of the apical brush border membrane. 70-kD FITC-dextran added apically to Myo5b KO intestinal explants accumulated in intracellular inclusions. Live imaging of Myo5b KO-derived enteroids confirmed the formation of inclusions from the apical membrane. Treatment of intestinal explants and enteroids with Dyngo resulted in accumulation of inclusions at the apical membrane. Inclusions in Myo5b KO enterocytes contained VAMP4 and Pacsin 2 (Syndapin 2). Myo5b;Pacsin 2 double-KO mice showed a significant decrease in inclusion formation. Our results suggest that apical bulk endocytosis in Myo5b KO enterocytes resembles activity-dependent bulk endocytosis, the primary mechanism for synaptic vesicle uptake during intense neuronal stimulation. Thus, apical bulk endocytosis mediates the formation of inclusions in neonatal Myo5b KO enterocytes.

Desmosomal cadherin association with Tctex-1 and cortactin-Arp2/3 drives perijunctional actin polymerization to promote keratinocyte delamination.

The epidermis is a multi-layered epithelium that serves as a barrier against water loss and environmental insults. Its morphogenesis occurs through a tightly regulated program of biochemical and architectural changes during which basal cells commit to differentiate and move towards the skin's surface. Here, we reveal an unexpected role for the vertebrate cadherin desmoglein 1 (Dsg1) in remodeling the actin cytoskeleton to promote the transit of basal cells into the suprabasal layer through a process of delamination, one mechanism of epidermal stratification. Actin remodeling requires the interaction of Dsg1 with the dynein light chain, Tctex-1 and the actin scaffolding protein, cortactin. We demonstrate that Tctex-1 ensures the correct membrane compartmentalization of Dsg1-containing desmosomes, allowing cortactin/Arp2/3-dependent perijunctional actin polymerization and decreasing tension at E-cadherin junctions to promote keratinocyte delamination. Moreover, Dsg1 is sufficient to enable simple epithelial cells to exit a monolayer to form a second layer, highlighting its morphogenetic potential.