Intravital Subcellular Microscopy of the Mammary Gland

The mammary gland constitutes a model par excellence for investigating epithelial functions, including tissue remodeling, cell polarity, and secretory ...

National Institutes of Health

University of Virginia

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.

NMII forms a contractile transcellular sarcomeric network to regulate apical cell junctions and tissue geometry.

Myosin transcellular networks regulate epithelial apical geometry.

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.

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

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. 

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

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.

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

Isoform-specific roles of NMII drive membrane remodeling in vivo.

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.

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

The remodeling of biological membranes is crucial for a vast number of cellular activities and is an inherently multiscale process in both time and space. Seminal work has provided important insights into nanometer-scale membrane deformations, and highlighted the remarkable variation and complexity in the underlying molecular machineries and mechanisms. However, how membranes are remodeled at the micron-scale, particularly in vivo, remains poorly understood. Here, we discuss how using regulated exocytosis of large (1.5-2.0 μm) membrane-bound secretory granules in the salivary gland of live mice as a model system, has provided evidence for the importance of the actomyosin cytoskeleton in micron-scale membrane remodeling in physiological conditions. We highlight some of these advances, and present mechanistic hypotheses for how the various biochemical and biophysical properties of distinct actomyosin networks may drive this process.

The Actomyosin Cytoskeleton Drives Micron-Scale Membrane Remodeling In Vivo Via the Generation of Mechanical Forces to Balance Membrane Tension Gradients.

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 postnatal formation. In addition, intravital subcellular microscopy revealed that reduced levels of Cdc42 affected membrane trafficking from and toward the plasma membrane, highlighting a novel role for Cdc42 in membrane remodeling through the negative regulation of selected endocytic pathways.

Cdc42 negatively regulates endocytosis during apical membrane maintenance in live animals.

Imaging subcellular processes in live animals is no longer a dream. The development of Intravital Subcellular Microscopy (ISMic) combined with the astounding repertoire of available mouse models makes it possible to investigate processes such as membrane trafficking in mammalian living tissues under native conditions. This has provided the unique opportunity to answer questions that cannot be otherwise addressed in reductionist model systems and to link cell biology to tissue pathophysiology.

Intravital microscopy in mammalian multicellular organisms.

The glycocalyx is a highly hydrated, glycoprotein-rich coat shrouding many eukaryotic and prokaryotic cells. The intestinal epithelial glycocalyx, comprising glycosylated transmembrane mucins, is part of the primary host-microbe interface and is essential for nutrient absorption. Its disruption has been implicated in numerous gastrointestinal diseases. Yet, due to challenges in preserving and visualizing its native organization, glycocalyx structure-function relationships remain unclear. Here, we characterize the nanoarchitecture of the murine enteric glycocalyx using freeze-etching and electron tomography. Micrometer-long mucin filaments emerge from microvillar-tips and, through zigzagged lateral interactions form a three-dimensional columnar network with a 30 nm mesh. Filament-termini converge into globular structures ~30 nm apart that are liquid-crystalline packed within a single plane. Finally, we assess glycocalyx deformability and porosity using intravital microscopy. We argue that the columnar network architecture and the liquid-crystalline packing of the filament termini allow the glycocalyx to function as a deformable size-exclusion filter of luminal contents.

Seham Ebrahim

Laboratory of Cellular and Molecular Biology,
Center for Cancer Research,
National Cancer Institute,
Laboratory of Cellular and Molecular Biology, Center for Cancer Research, National Cancer Institute

National Institutes of Health

Intravital Subcellular Microscopy of the Mammary Gland

Seham Ebrahim

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}Laboratory of Cellular and Molecular Biology,Center for Cancer Research,National Cancer Institute,Laboratory of Cellular and Molecular Biology, Center for Cancer Research, National Cancer Institute

National Institutes of Health

Intravital Subcellular Microscopy of the Mammary Gland

Laboratory of Cellular and Molecular Biology,
Center for Cancer Research,
National Cancer Institute,
Laboratory of Cellular and Molecular Biology, Center for Cancer Research, National Cancer Institute