Seham Ebrahim

NMII Forms a Contractile Transcellular Sarcomeric Network to Regulate Apical Cell Junctions and Tissue Geometry

Nonmuscle myosin II (NMII) is thought to be the master integrator of force within epithelial apical junctions, mediating epithelial tissue morphogenesis and tensional homeostasis. Mutations in NMII are associated with a number of diseases due to failures in cell-cell adhesion. However, the organization and the precise mechanism by which NMII generates and responds to tension along the intercellular junctional line are still not known. We discovered that periodic assemblies of bipolar NMII filaments interlace with perijunctional actin and α-actinin to form a continuous belt of muscle-like sarcomeric units (∼400-600 nm) around each epithelial cell. Remarkably, the sarcomeres of adjacent cells are precisely paired across the junctional line, forming an integrated, transcellular contractile network. The contraction/relaxation of paired sarcomeres concomitantly impacts changes in apical cell shape and tissue geometry. We show differential distribution of NMII isoforms across heterotypic junctions and evidence for compensation between isoforms. Our results provide a model for how NMII force generation is effected along the junctional perimeter of each cell and communicated across neighboring cells in the epithelial organization. The sarcomeric network also provides a well-defined target to investigate the multiple roles of NMII in junctional homeostasis as well as in development and disease.

TMC1 and TMC2 Localize at the Site of Mechanotransduction in Mammalian Inner Ear Hair Cell Stereocilia.

Mechanosensitive ion channels at stereocilia tips mediate mechanoelectrical transduction (MET) in inner ear sensory hair cells. Transmembrane channel-like 1 and 2 (TMC1 and TMC2) are essential for MET and are hypothesized to be components of the MET complex, but evidence for their predicted spatiotemporal localization in stereocilia is lacking. Here, we determine the stereocilia localization of the TMC proteins in mice expressing TMC1-mCherry and TMC2-AcGFP. Functionality of the tagged proteins was verified by transgenic rescue of MET currents and hearing in Tmc1(Δ/Δ);Tmc2(Δ/Δ) mice. TMC1-mCherry and TMC2-AcGFP localize along the length of immature stereocilia. However, as hair cells develop, the two proteins localize predominantly to stereocilia tips. Both TMCs are absent from the tips of the tallest stereocilia, where MET activity is not detectable. This distribution was confirmed for the endogenous proteins by immunofluorescence. These data are consistent with TMC1 and TMC2 being components of the stereocilia MET channel complex.

Stereocilia-staircase spacing is influenced by myosin III motors and their cargos espin-1 and espin-like

Hair cells tightly control the dimensions of their stereocilia, which are actin-rich protrusions with graded heights that mediate mechanotransduction in the inner ear. Two members of the myosin-III family, MYO3A and MYO3B, are thought to regulate stereocilia length by transporting cargos that control actin polymerization at stereocilia tips. We show that eliminating espin-1 (ESPN-1), an isoform of ESPN and a myosin-III cargo, dramatically alters the slope of the stereocilia staircase in a subset of hair cells. Furthermore, we show that espin-like (ESPNL), primarily present in developing stereocilia, is also a myosin-III cargo and is essential for normal hearing. ESPN-1 and ESPNL each bind MYO3A and MYO3B, but differentially influence how the two motors function. Consequently, functional properties of different motor-cargo combinations differentially affect molecular transport and the length of actin protrusions. This mechanism is used by hair cells to establish the required range of stereocilia lengths within a single cell.

Concerted actions of distinct nonmuscle myosin II isoforms drive intracellular membrane remodeling in live animals

Membrane remodeling plays a fundamental role during a variety of biological events. However, the dynamics and the molecular mechanisms regulating this process within cells in mammalian tissues in situ remain largely unknown. In this study, we use intravital subcellular microscopy in live mice to study the role of the actomyosin cytoskeleton in driving the remodeling of membranes of large secretory granules, which are integrated into the plasma membrane during regulated exocytosis. We show that two isoforms of nonmuscle myosin II, NMIIA and NMIIB, control distinct steps of the integration process. Furthermore, we find that F-actin is not essential for the recruitment of NMII to the secretory granules but plays a key role in the assembly and activation of NMII into contractile filaments. Our data support a dual role for the actomyosin cytoskeleton in providing the mechanical forces required to remodel the lipid bilayer and serving as a scaffold to recruit key regulatory molecules.

Maturation arrest in early postnatal sensory receptors by deletion of the miR-183/96/182 cluster in mouse

Significance MicroRNAs (miRNAs) are small noncoding RNAs that regulate gene expression posttranscriptionally. The evolutionarily conserved miR-183/96/182 cluster, consisting of three related miRNAs, is highly expressed in maturing sensory receptor cells. However, its role in the functional maturation of sensory receptors has not been adequately addressed due to the lack of appropriate in vivo models. We show that deletion of miR-183/96/182 in mice leads to severe deficits in vision, hearing, balance, and smell. These deficits arise from defects in the timing and completion of terminal differentiation in sensory receptor cells associated with dysregulation of networks of genes involved in key processes, such as chromatin remolding and ciliogenesis. Thus, the miR-183/96/182 cluster has an essential role for the maturation of sensory receptors. The polycistronic miR-183/96/182 cluster is preferentially and abundantly expressed in terminally differentiating sensory epithelia. To clarify its roles in the terminal differentiation of sensory receptors in vivo, we deleted the entire gene cluster in mouse germline through homologous recombination. The miR-183/96/182 null mice display impairment of the visual, auditory, vestibular, and olfactory systems, attributable to profound defects in sensory receptor terminal differentiation. Maturation of sensory receptor precursors is delayed, and they never attain a fully differentiated state. In the retina, delay in up-regulation of key photoreceptor genes underlies delayed outer segment elongation and possibly mispositioning of cone nuclei in the retina. Incomplete maturation of photoreceptors is followed shortly afterward by early-onset degeneration. Cell biologic and transcriptome analyses implicate dysregulation of ciliogenesis, nuclear translocation, and an epigenetic mechanism that may control timing of terminal differentiation in developing photoreceptors. In both the organ of Corti and the vestibular organ, impaired terminal differentiation manifests as immature stereocilia and kinocilia on the apical surface of hair cells. Our study thus establishes a dedicated role of the miR-183/96/182 cluster in driving the terminal differentiation of multiple sensory receptor cells.

Myosin transcellular networks regulate epithelial apical geometry

To fulfill their numerous tissue- and organ-specific roles, epithelial sheets rely on specialized architecture and specific mechanical properties, ranging from the exquisitely patterned, force-balanced sensory epithelia of the inner ear, to the highly dynamic and convoluted epithelia lining the villi and crypts of the intestinal tract. Both the morphology and mechanical characteristics of epithelial sheets are emergent properties of their constituent cells; subtle shrinking of the apical boundaries of epithelial cells can lead to larger-scale changes in packing geometry, even causing dramatic bending of epithelial sheets. 1 However, the molecular and structural underpinnings for the apical constriction of epithelial cells are not fully understood. Non-muscle myosin II (NMII) has been implicated in driving a “purse string”-like contraction of the circumferential actin belt that encircles the inner surface of epithelial cells. 2,3 Current models depict NMII filaments randomly distributed along the circumferential actin belt, 4 but it is unclear how the activities of individual NMII filaments are integrated and coordinated to generate and transmit force across the epithelium. Understanding these processes is crucial to understanding epithelial dynamics during development, as well as the post-developmental maintenance of epithelial architecture and tensional homeostasis. In a recent study we focused on elucidating the distribution, organization, and role of NMII isoforms within epithelial apical junctions using the organ of Corti, one of the most striking examples of mammalian epithelial patterning, as a model system. 5 Seeking to investigate the effects of NMII inhibition on apical junctions, we treated explant cultures of the organ of Corti with the NMIIspecific inhibitor blebbistatin. 6 This induced changes in the epithelial apical surface at both a cellular and tissue level. Morphometric quantification showed an increase in junctional-length, a corresponding increase in apical cell-surface area, and a deformation of apical cell shape across the tissue, resulting in an overall expansion of the epithelial sheet. Strikingly, the changes were completely reversed upon washout of the blebbistatin. Our observations suggested that apical perimeters of epithelial cells are dynamically maintained under tension by NMII within the circumferential junctional actomyosin belt. With data supporting a role for NMII in influencing epithelial apical junctions, we next determined the pattern of distribution of each NMII paralog, NMIIA, IIB, and IIC, along the cell perimeter. Immunofluorescence of NMIIB and NMIIC showed a striking distribution as regularly spaced puncta associated with perijunctional actin. Measurements of relative fluorescence intensity revealed low actin density at NMII fluorescence puncta and higher actin density between them, resembling the sarcomeric striations in myofibrils. This finding prompted us to test for the presence of another hallmark component of muscle sarcomeres, the actin crosslinker α-actinin. Immunofluorescence confirmed that α-actinin1 is in fact present along the junctional line in a periodic distribution, alternating with NMII and coinciding with actin, just like in sarcomeres. Another essential feature of sarcomeric organization is the orientation of bipolar myosin filaments parallel to actin filaments. To determine the polarity and orientation of NMII filaments along the apical perimeter of epithelial cells, we exogenously expressed, in organ of Corti cultures, NMIIC tagged with different fluorophores at the N terminus and C terminus. We also labeled tissue from an NMIIC–GFP transgenic mouse (GFP-tag at the C terminus), with antibody against the NMIIC N terminus. These experiments confirmed that bipolar NMII filaments organize as arrays

Corrigendum: Stereocilia-staircase spacing is influenced by myosin III motors and their cargos espin-1 and espin-like

This corrects the article DOI: 10.1038/ncomms10833.

Isoform-specific roles of NMII drive membrane remodeling in vivo

Lipid-based membranes are self-assembling, multicomponent molecular layers that surround cells and organelles. They are made up of a variety of distinct lipids and transmembrane proteins, and also associate peripherally with proteins such as scaffolds that sense, induce or stabilize membrane-curvature, and/or molecular motors and cytoskeletal proteins that exert forces. Protein-induced dynamic changes in membrane curvature are essential for processes ranging from cell motility and cytokinesis, to neuroplasticity and intracellular trafficking. Accordingly, defects in membrane remodeling are associated withmany human pathologies including neuromuscular and autoimmune diseases and cancers. Understanding how the cooperative contributions of multiple molecular components drive membrane remodeling is therefore a fundamental challenge in biology. Most studies aimed at addressing this challenge use reductionist model systems such as model membranes, cell culture, or cultured organs. While these approaches provide important insights, they do not faithfully recapitulate the complex environments and machineries required for membrane remodeling in live multicellular organisms (in vivo), and are thus limited in the extent of their physiological translatability. To visualize and characterize membrane remodeling in vivo, we developed intravital subcellular microscopy (ISMic), a light microscopy-based technique tailored to image intracellular organelles in live rodents. As our model system to study membrane dynamics we used the process of regulated exocytosis in mouse salivary glands. During this process (Fig. 1), large (»1.5 mm) membrane-bound salivary granules (SGs) containing proteinous cargoes fuse with the canalicular-apical plasma membrane (APM) of salivary acinar cells upon GPCR stimulation. The consequent opening of the fusion pore then allows release of the granular contents into the canaliculi, and the SG membrane slowly integrates into the APM over »60 seconds. Since the diameter of acinar canaliculi is in the range of 0.3 mm, almost an order of magnitude smaller than SG diameter, the complete integration of the SG membrane into the APM is energetically unfavorable, raising a number of questions: What external mechanical forces drive the integration of SG membranes into the APM? What proteins supply and regulate these forces? And what is the spatiotemporal order in which these force-generating proteins are recruited? Investigating the role of the cytoskeleton in SG integration in vivo, we previously showed that pharmacological inhibition of either F-actin assembly or non-muscle myosin II (NMII) motor activity impeded integration, indicating that the acto-myosin cytoskeleton provides the driving force required to complete this process. More recently, using new mouse models, we sought to further advance the molecular characterization of SG integration by investigating: 1) the specific role(s) played by distinct NMII isoforms, and 2) the mechanisms of their recruitment and activation. Usingmice expressing fluorescently-taggedNMII isoforms, we first determined that two isoforms of NMII, namely NMIIA and NMIIB, localize to fused SGs. Next, using ISMic in live mice expressing a reporter for F-actin (LifeAct) in addition to either fluorescently-tagged NMII isoform, we discovered that both isoforms appear on fused SGs seconds after F-actin. While this initially suggested that F-actin may be recruiting NMII to the SG surface, we were surprised to find that pharmacological disruption of F-actin assembly did not affect the localization of either NMIIA or NMIIB to fused SGs. This finding introduces the fascinating prospect of a yet-to-be-identified, actin-independent mode of NMII recruitment (Fig. 1B). To examine the specific roles of NMIIA and NMIIB in the integration process in acinar cells, we used transgenic mice in which genes encoding either NMIIA or NMIIB or both NMIIA/NMIIB were floxed, and knockdown was achieved by in vivo transfection of Cre-recombinase. Analyzing the kinetics of granule integration in each knockdown we found that: 1) In the absence of NMIIA, the kinetics of initial integration remained unaffected until SGs reached 50–60% of their initial diameter, at which point integration was stalled; 2) In NMIIB knockdown cells, SGs underwent an initial increase in diameter, followed by a delayed (but complete) integration; and 3) ablation of both NMIIA and NMIIB blocked integration in all but a small percentage of SGs, raising the possibility of an additional, myosin-independent mechanism. Based on these data, we propose the following model where NMIIA and NMIIB play separate roles during the integration process, consistent with their different enzymatic properties: First NMIIB stabilizes the membranes of fused SGs and, through slow contractions and cross-linking, drives the initial process of integration.

Cdc42 negatively regulates endocytosis during apical plasma membrane maintenance and development in mouse tubular organs in vivo

Lumen establishment and maintenance are fundamental for tubular organs physiological functions. Most of the studies investigating the mechanisms regulating this process have been carried out in cell cultures or in smaller organisms, whereas little has been done in mammalian model systems in vivo. Here we used the salivary glands of live mice to examine the role of the small GTPase Cdc42 in the regulation of the homeostasis of the intercellular canaliculi, a specialized apical domain of the acinar cells, where protein and fluid secretion occur. Depletion of Cdc42 in adult mice induced a significant expansion of the apical canaliculi, whereas depletion at late embryonic stages resulted in a complete inhibition of their post-natal formation. In addition, intravital subcellular microscopy revealed that reduced levels of Cdc42 affected membrane trafficking from and towards the plasma membrane, highlighting a novel role for Cdc42 in membrane remodeling through the negative regulation of selected endocytic pathways.

Variable number of TMC1-dependent mechanotransducer channels underlie tonotopic conductance gradients in the cochlea

Functional mechanoelectrical transduction (MET) channels of cochlear hair cells require the presence of transmembrane channel-like protein isoforms TMC1 or TMC2. We show that TMCs are required for normal stereociliary bundle development and distinctively influence channel properties. TMC1-dependent channels have larger single-channel conductance and in outer hair cells (OHCs) support a tonotopic apex-to-base conductance gradient. Each MET channel complex exhibits multiple conductance states in ~50 pS increments, basal MET channels having more large-conductance levels. Using mice expressing fluorescently tagged TMCs, we show a three-fold increase in number of TMC1 molecules per stereocilium tip from cochlear apex to base, mirroring the channel conductance gradient in OHCs. Single-molecule photobleaching indicates the number of TMC1 molecules per MET complex changes from ~8 at the apex to ~20 at base. The results suggest there are varying numbers of channels per MET complex, each requiring multiple TMC1 molecules, and together operating in a coordinated or cooperative manner.Mechanoelectrical transduction channel (MET) current found in stereocilia of hair cells matures over the first postnatal week. Here the authors look at the contribution of transmembrane channel-like protein 1 and 2 (TMC1 and TMC2) to MET current during development of tonotopic gradients.