Intestinal brush border assembly driven by protocadherin-based intermicrovillar adhesion.

Transporting epithelial cells build apical microvilli to increase membrane surface area and enhance absorptive capacity. The intestinal brush border provides an elaborate example with tightly packed microvilli that function in nutrient absorption and host defense. Although the brush border is essential for physiological homeostasis, its assembly is poorly understood. We found that brush border assembly is driven by the formation of Ca(2+)-dependent adhesion links between adjacent microvilli. Intermicrovillar links are composed of protocadherin-24 and mucin-like protocadherin, which target to microvillar tips and interact to form a trans-heterophilic complex. The cytoplasmic domains of microvillar protocadherins interact with the scaffolding protein, harmonin, and myosin-7b, which promote localization to microvillar tips. Finally, a mouse model of Usher syndrome lacking harmonin exhibits microvillar protocadherin mislocalization and severe defects in brush border morphology. These data reveal an adhesion-based mechanism for brush border assembly and illuminate the basis of intestinal pathology in patients with Usher syndrome. PAPERFLICK:

Gpr126 functions in Schwann cells to control differentiation and myelination via G-protein activation.

The myelin sheath surrounding axons ensures that nerve impulses travel quickly and efficiently, allowing for the proper function of the vertebrate nervous system. We previously showed that the adhesion G-protein-coupled receptor (aGPCR) Gpr126 is essential for peripheral nervous system myelination, although the molecular mechanisms by which Gpr126 functions were incompletely understood. aGPCRs are a significantly understudied protein class, and it was unknown whether Gpr126 couples to G-proteins. Here, we analyze Dhh(Cre);Gpr126(fl/fl) conditional mutants, and show that Gpr126 functions in Schwann cells (SCs) for radial sorting of axons and myelination. Furthermore, we demonstrate that elevation of cAMP levels or protein kinase A activation suppresses myelin defects in Gpr126 mouse mutants and that cAMP levels are reduced in conditional Gpr126 mutant peripheral nerve. Finally, we show that GPR126 directly increases cAMP by coupling to heterotrimeric G-proteins. Together, these data support a model in which Gpr126 functions in SCs for proper development and myelination and provide evidence that these functions are mediated via G-protein-signaling pathways.

Proteomic analysis of the enterocyte brush border.

The brush border domain at the apex of intestinal epithelial cells is the primary site of nutrient absorption in the intestinal tract and the primary surface of interaction with microbes that reside in the lumen. Because the brush border is positioned at such a critical physiological interface, we set out to create a comprehensive list of the proteins that reside in this domain using shotgun mass spectrometry. The resulting proteome contains 646 proteins with diverse functions. In addition to the expected collection of nutrient processing and transport components, we also identified molecules expected to function in the regulation of actin dynamics, membrane bending, and extracellular adhesion. These results provide a foundation for future studies aimed at defining the molecular mechanisms underpinning brush border assembly and function.

The synovial sarcoma SYT-SSX2 oncogene remodels the cytoskeleton through activation of the ephrin pathway.

Synovial sarcoma is a soft tissue cancer associated with a recurrent t(X:18) translocation that generates one of two fusion proteins, SYT-SSX1 or SYT-SSX2. In this study, we demonstrate that SYT-SSX2 is a unique oncogene. Rather than confer enhanced proliferation on its target cells, SYT-SSX2 instead causes a profound alteration of their architecture. This aberrant morphology included elongation of the cell body and formation of neurite-like extensions. We also observed that cells transduced with SYT-SSX2 often repulsed one another. Notably, cell repulsion is a known component of ephrin signaling. Further analysis of SYT-SSX2-infected cells revealed significant increases in the expression and activation of Eph/ephrin pathway components. On blockade of EphB2 signaling SYT-SSX2 infectants demonstrated significant reversion of the aberrant cytoskeletal phenotype. In addition, we discovered, in parallel, that SYT-SSX2 induced stabilization of the microtubule network accompanied by accumulation of detyrosinated Glu tubulin and nocodazole resistance. Glu tubulin regulation was independent of ephrin signaling. The clinical relevance of these studies was confirmed by abundant expression of both EphB2 and Glu tubulin in SYT-SSX2-positive synovial sarcoma tissues. These results indicate that SYT-SSX2 exerts part of its oncogenic effect by altering cytoskeletal architecture in an Eph-dependent manner and cytoskeletal stability through a concurrent and distinct pathway.

Expression and localization of myosin-1d in the developing nervous system.

Myosin-1d is a monomeric actin-based motor found in a wide range of tissues, but highly expressed in the nervous system. Previous microarray studies suggest that myosin-1d is found in oligodendrocytes where transcripts are upregulated during the maturation of these cells. Myosin-1d was also identified as a component of myelin-containing subcellular fractions in proteomic studies and mutations in MYO1D have been linked to autism. Despite the potential implications of these previous studies, there is little information on the expression and localization of myosin-1d in the developing nervous system. Therefore, we analyzed myosin-1d expression patterns in the peripheral and central nervous systems during postnatal development. In mouse sciatic nerve, myosin-1d is expressed along the axon and in the ensheathing myelin compartment. Analysis of mouse cerebellum prior to myelination at day 3 reveals that myosin-1d is present in the Purkinje cell layer, granule cell layer, and region of the cerebellar nuclei. Upon the onset of myelination, myosin-1d enrichment expands along axonal tracts, while still present in the Purkinje and granule cell layers. However, myosin-1d was undetectable in oligodendrocyte progenitor cells at early and late time points. We also show that myosin-1d interacts and is co-expressed with aspartoacylase, an enzyme that plays a key role in fatty acid synthesis throughout the nervous system. Together, these studies provide a foundation for understanding the role of myosin-1d in neurodevelopment and neurological disorders.

Differential localization and dynamics of class I myosins in the enterocyte microvillus.

Epithelial cells lining the intestinal tract build an apical array of microvilli known as the brush border. Each microvillus is a cylindrical membrane protrusion that is linked to a supporting actin bundle by myosin-1a (Myo1a). Mice lacking Myo1a demonstrate no overt physiological symptoms, suggesting that other myosins may compensate for the loss of Myo1a in these animals. To investigate changes in the microvillar myosin population that may limit the Myo1a KO phenotype, we performed proteomic analysis on WT and Myo1a KO brush borders. These studies revealed that WT brush borders also contain the short-tailed class I myosin, myosin-1d (Myo1d). Myo1d localizes to the terminal web and striking puncta at the tips of microvilli. In the absence of Myo1a, Myo1d peptide counts increase twofold; this motor also redistributes along the length of microvilli, into compartments normally occupied by Myo1a. FRAP studies demonstrate that Myo1a is less dynamic than Myo1d, providing a mechanistic explanation for the observed differential localization. These data suggest that Myo1d may be the primary compensating class I myosin in the Myo1a KO model; they also suggest that dynamics govern the localization and function of different yet closely related myosins that target common actin structures.