The sarcomere is the contractile unit within cardiomyocytes driving heart muscle contraction. We sought to test the mechanisms regulating actin and myosin filament assembly during sarcomere formation. Therefore, we developed an assay using human cardiomyocytes to monitor sarcomere assembly. We report a population of muscle stress fibers, similar to actin arcs in non-muscle cells, which are essential sarcomere precursors. We show sarcomeric actin filaments arise directly from muscle stress fibers. This requires formins (e.g., FHOD3), non-muscle myosin IIA and non-muscle myosin IIB. Furthermore, we show short cardiac myosin II filaments grow to form ~1.5 μm long filaments that then ‘stitch’ together to form the stack of filaments at the core of the sarcomere (i.e., the A-band). A-band assembly is dependent on the proper organization of actin filaments and, as such, is also dependent on FHOD3 and myosin IIB. We use this experimental paradigm to present evidence for a unifying model of sarcomere assembly.

Clinical evidence supports a role for the extracellular matrix (ECM) in cancer risk and prognosis across multiple tumor types, and numerous studies have demonstrated that individual ECM components impact key hallmarks of tumor progression (e.g., proliferation, migration, angiogenesis). However, the ECM is a complex network of fibrillar proteins, glycoproteins, and proteoglycans that undergoes dramatic changes in composition and organization during tumor development. In this review, we will highlight how engineering approaches can be used to examine the impact of changes in tissue architecture, ECM composition (i.e., identity and levels of individual ECM components), and cellular-and tissue-level mechanics on tumor progression. In addition, we will discuss recently developed methods to model the ECM that have not yet been applied to the study of cancer.

Calcium signals via calmodulin, myosin light chain kinase, and nonmuscle myosin II to mediate neuroepithelial apical-basal cell length during zebrafish brain morphogenesis.

No abstract

Although microtubules are known for dynamic instability, the dynamicity is considered to be tightly controlled to support a variety of cellular processes. Yet diverse evidence suggests that this is not applicable to Chlamydomonas, a biflagellate fresh water green alga, but intense autofluorescence from photosynthesis pigments has hindered the investigation. By expressing a bright fluorescent reporter protein at the endogenous level, we demonstrate in real time discreet sweeping changes in algal microtubules elicited by rises of intracellular H+ and Na+. These results from this model organism with characteristics of animal and plant cells provide novel explanations regarding how pH may drive cellular processes; how plants may respond to, and perhaps sense stresses; and how organisms with a similar sensitive cytoskeleton may be susceptible to environmental changes.

Recent evidence supports the fimbriae of the fallopian tube as one origin site for high-grade serous ovarian cancer (HGSOC). The progression of many solid tumors is accompanied by changes in the microenvironment, including alterations of the extracellular matrix (ECM). Therefore, we sought to determine the ECM composition of the benign fallopian tube and changes associated with serous tubal intraepithelial carcinomas (STICs), precursors of HGSOC. The ECM composition of benign human fallopian tube was first defined from a meta-analysis of published proteomic datasets that identified 190 ECM proteins. We then conducted de novo proteomics using ECM enrichment and identified 88 proteins, 7 of which were not identified in prior studies (COL2A1, COL4A5, COL16A1, elastin, LAMA5, annexin A2, and PAI1). To enable future in vitro studies, we investigated the levels and localization of ECM components included in tissue-engineered models (type I, III, and IV collagens, fibronectin, laminin, versican, perlecan, and hyaluronic acid) using multispectral immunohistochemical staining of fimbriae from patients with benign conditions or STICs. Quantification revealed an increase in stromal fibronectin and a decrease in epithelial versican in STICs. Our results provide an in-depth picture of the ECM in the benign fallopian tube and identified ECM changes that accompany STIC formation. (J Histochem Cytochem XX: XXX–XXX, XXXX)

The folding of epithelial tissues is crucial for development of three-dimensional structure and function. Understanding this process can assist in determining the etiology of developmental disease and engineering of tissues for the future of regenerative medicine. Folding of epithelial tissues towards the apical surface has long been studied, but the molecular mechanisms that mediate epithelial folding towards the basal surface are just emerging. Here, we utilize zebrafish neuroepithelium to identify mechanisms that mediate basal tissue folding to form the highly conserved embryonic midbrain-hindbrain boundary. Live imaging revealed Wnt5b as a mediator of anisotropic epithelial cell shape, both apically and basally. In addition, we uncovered a Wnt5b-mediated mechanism for specific regulation of basal anisotropic cell shape that is microtubule dependent and likely to involve JNK signaling. We propose a model in which a single morphogen can differentially regulate apical versus basal cell shape during tissue morphogenesis.

The sarcomere is the contractile unit that drives muscle contraction. Despite its importance, little is understood about how a disordered acto-myosin distribution converts into an ordered contractile array during sarcomere assembly. Here, we take advantage of a sarcomere assembly assay we developed using human induced pluripotent stem cell derived cardiomycytes to image the formation of sarcomeres, using live-cell high resolution microscopy. Our data show that a population of muscle specific stress fibers (MSFs) are essential sarcomere precursors. Interestingly, MSFs are formed at the leading edge of cells, undergo retrograde flow, and transition into sarcomere-containing myofibrils on the dorsal surface of cardiomyocytes. This is in direct contradiction to a recent report claiming sarcomeres are formed from adhesions on the ventral surface of cardiomyocytes. We have been able to recapitulate this other group's published experiments and definitively show that sarcomeres are not forming from the bottom of the cells or streaming out of adhesions. Instead, our 3D microscopy data shows that this group was imaging sarcomeres which were already formed on the dorsal surface and were traveling to the ventral surface of the cells. After this important clarification, we used our assay to show that the transition of MSFs to sarcomere-containing myofibrils requires formin-mediated actin polymerization and the non-muscle myosin IIA and myosin IIB. We conclude that sarcomeres form by a "templating" mechanism similar to that originally postulated by Howard Holtzer >30 years ago. Furthermore, our data show short β cardiac myosin II filaments are themselves templated by "non-muscle" myosin II filaments. Subsequently, the short β cardiac myosin II filaments grow to form ~1.5 μm long filaments that then “stitch” together to form the stack of filaments at the core of the sarcomere (i.e., the A-band). Taken together, our data show that the differentiation of cardiomyocytes from stem cells is a powerful tool for dissecting the mechanisms controlling sarcomere assembly.