The formation of the primitive heart tube from cardiomyocytes and endocardial cells is a key event in mammalian development. Previous studies suggested that cardiomyocytes and endocardial cells segregate from a shared cardiac progenitor around the onset of gastrulation, yet their lineage relationship with other mesodermal tissues remains unclear. Using retrospective and prospective clonal analyses in mouse embryos, we traced cardiomyocyte and endocardial progenitors from the primitive streak to the heart tube. Our results identify two independent mesodermal populations specified around gastrulation onset. While each of these populations is unipotent in producing cardiomyocytes or endocardium, they retain multipotency and contribute to different subsets of non-cardiac mesoderm. Nonetheless, live imaging identifies simultaneous ingression and intermingling of these two mesodermal lineages in the primitive streak, showing their coordinated specification and migration. The proposed model for cardiac progenitor specification will help understanding the origins of congenital heart diseases and designing tissue engineering strategies.

Class-18 myosins are most closely related to conventional class-2 nonmuscle myosins (NM2). Surprisingly, the purified head domains of Drosophila, mouse, and human myosin 18A (M18A) lack actin-activated ATPase activity and the ability to translocate actin filaments, suggesting that the functions of M18A in vivo do not depend on intrinsic motor activity. M18A has the longest coiled coil of any myosin outside of the class-2 myosins, suggesting that it might form bipolar filaments similar to conventional myosins. To address this possibility, we expressed and purified full-length mouse M18A using the baculovirus/Sf9 system. M18A did not form large bipolar filaments under any of the conditions tested. Instead, M18A formed an ∼65-nm-long bipolar structure with two heads at each end. Importantly, when NM2 was polymerized in the presence of M18A, the two myosins formed mixed bipolar filaments, as evidenced by cosedimentation, electron microscopy, and single-molecule imaging. Moreover, super-resolution imaging of NM2 and M18A using fluorescently tagged proteins and immunostaining of endogenous proteins showed that NM2 and M18A are present together within individual filaments inside living cells. Together, our in vitro and live-cell imaging data argue strongly that M18A coassembles with NM2 into mixed bipolar filaments. M18A could regulate the biophysical properties of these filaments and, by virtue of its extra N- and C-terminal domains, determine the localization and/or molecular interactions of the filaments. Given the numerous, fundamental cellular and developmental roles attributed to NM2, our results have far-reaching biological implications.

During transcription, RNA Polymerase II (RNAPII) is spatially organised within the nucleus into clusters that correlate with transcription activity. While this is a hallmark of genome regulation in mammalian cells, the mechanisms concerning the assembly, organisation and stability remain unknown. Here, we have used combination of single molecule imaging and genomic approaches to explore the role of nuclear myosin VI (MVI) in the nanoscale organisation of RNAPII. We reveal that MVI in the nucleus acts as the molecular anchor that holds RNAPII in high density clusters. Perturbation of MVI leads to the disruption of RNAPII localisation, chromatin organisation and subsequently a decrease in gene expression. Overall, we uncover the fundamental role of MVI in the spatial regulation of gene expression.

The distinct electrical properties of axonal and dendritic membranes are largely a result of specific transport of vesicle-bound membrane proteins to each compartment. How this specificity arises is unclear because kinesin motors that transport vesicles cannot autonomously distinguish dendritically projecting microtubules from those projecting axonally. We hypothesized that interaction with a second motor might enable vesicles containing dendritic proteins to preferentially associate with dendritically projecting microtubules and avoid those that project to the axon. Here we show that in rat cortical neurons, localization of several distinct transmembrane proteins to dendrites is dependent on specific myosin motors and an intact actin network. Moreover, fusion with a myosin-binding domain from Melanophilin targeted Channelrhodopsin-2 specifically to the somatodendritic compartment of neurons in mice in vivo. Together, our results suggest that dendritic transmembrane proteins direct the vesicles in which they are transported to avoid the axonal compartment through interaction with myosin motors.

Nancy E. Beckage is widely recognized for her pioneering work in the field of insect host-parasitoid interactions beginning with endocrine influences of the tobacco hornworm, Manduca sexta, host and its parasitoid wasp Apanteles congregatus (now Cotesia congregata) on each other’s development. Moreover, her studies show that the polydnavirus carried by the parasitoid wasp not only protects the parasitoid from the host’s immune defenses, but also is responsible for some of the developmental effects of parasitism. Nancy was a highly regarded mentor of both undergraduate and graduate students and more widely of women students and colleagues in entomology. Her service both to her particular area and to entomology in general through participation on federal grant review panels and in the governance of the Entomological Society of America, organization of symposia at both national and international meetings, and editorship of several different journal issues and of several books, is legendary. She has left behind a lasting legacy of increased understanding of multilevel endocrine and physiological interactions among insects and other organisms and a strong network of interacting scientists and colleagues in her area of entomology.

Genetically-encoded fluorescent biosensors have revolutionized our understanding of complex systems by permitting the in situ observation of chemical activities. However, only a comparatively small set of chemical activities can be monitored, largely due to the need to identify protein domains that undergo conformational and/or association changes in response to a stimulus. Here, we present a strategy that can convert ’simple’ affinity binders such as nanobodies into biosensors for their innate targets by introducing a peptide sequence that competes for the binding site. We demonstrate proof-of-concept implementations of this ’NanoBlock’ design, developing sensors based on the ALFA nanobody and on the PDZ domain of Erbin. We show that these sensors can reliably detect their targets in vitro, in mammalian cells, and as part of fluorescence-activated cell sorting (FACS) experiments. In doing so, our strategy offers a way to strongly expand the range of cellular processes that can be probed using fluorescent biosensors.

Nano-resolution fluorescence electron microscopy (nano-fEM) pinpoints the location of individual proteins in electron micrographs. Plastic sections are first imaged using a super-resolution fluorescence microscope and then imaged on an electron microscope. The two images are superimposed to correlate the position of labeled proteins relative to subcellular structures. Here, we describe the method in detail and present five technical advancements: the use of uranyl acetate during the freeze-substitution to enhance the contrast of tissues and reduce the loss of fluorescence, the use of ground-state depletion instead of photoactivation for temporal control of fluorescence, the use of organic fluorophores instead of fluorescent proteins to obtain brighter fluorescence signals, the use of tissue culture cells to broaden the utility of the method, and the use of a transmission electron microscope to achieve sharper images of ultrastructure.

A primary cilium is a thin membrane-bound extension off a cell surface that contains receptors for perceiving and transmitting signals that modulate cell state and activity. While many cell types have a primary cilium, little is known about primary cilia in the brain, where they are less accessible than cilia on cultured cells or epithelial tissues and protrude from cell bodies into a deep, dense network of glial and neuronal processes. Here, we investigated cilia frequency, internal structure, shape, and position in large, high-resolution transmission electron microscopy volumes of mouse primary visual cortex. Cilia extended from the cell bodies of nearly all excitatory and inhibitory neurons, astrocytes, and oligodendrocyte precursor cells (OPCs), but were absent from oligodendrocytes and microglia. Structural comparisons revealed that the membrane structure at the base of the cilium and the microtubule organization differed between neurons and glia. OPC cilia were distinct in that they were the shortest and contained pervasive internal vesicles only occasionally observed in neuron and astrocyte cilia. Investigating cilia-proximal features revealed that many cilia were directly adjacent to synapses, suggesting cilia are well poised to encounter locally released signaling molecules. The internal anatomy, including microtubule changes and centriole location, defined key structural features including cilium placement and shape. Together, the anatomical insights both within and around neuron and glia cilia provide new insights into cilia formation and function across cell types in the brain.

Retinal Müller glia function as injury-induced stem-like cells in zebrafish but not mammals. However, insights gleaned from zebrafish have been applied to stimulate nascent regenerative responses in the mammalian retina. For instance, microglia/macrophages regulate Müller glia stem cell activity in the chick, zebrafish, and mouse. We previously showed that post-injury immunosuppression by the glucocorticoid dexamethasone accelerated retinal regeneration kinetics in zebrafish. Similarly, microglia ablation enhances regenerative outcomes in the mouse retina. Targeted immunomodulation of microglia reactivity may therefore enhance the regenerative potential of Müller glia for therapeutic purposes. Here, we investigated potential mechanisms by which post-injury dexamethasone accelerates retinal regeneration kinetics, and the effects of dendrimer-based targeting of dexamethasone to reactive microglia. Intravital time-lapse imaging revealed that post-injury dexamethasone inhibited microglia reactivity. The dendrimer-conjugated formulation: (1) decreased dexamethasone-associated systemic toxicity, (2) targeted dexamethasone to reactive microglia, and (3) improved the regeneration enhancing effects of immunosuppression by increasing stem/progenitor proliferation rates. Lastly, we show that the gene rnf2 is required for the enhanced regeneration effect of D-Dex. These data support the use of dendrimer-based targeting of reactive immune cells to reduce toxicity and enhance the regeneration promoting effects of immunosuppressants in the retina.

Cell adhesions to the extracellular matrix (ECM) are necessary for morphogenesis, immunity, and wound healing. Focal adhesions are multifunctional organelles that mediate cell-ECM adhesion, force transmission, cytoskeletal regulation and signaling. Focal adhesions consist of a complex network of trans-plasma-membrane integrins and cytoplasmic proteins that form a <200-nm plaque linking the ECM to the actin cytoskeleton. The complexity of focal adhesion composition and dynamics implicate an intricate molecular machine. However, focal adhesion molecular architecture remains unknown. Here we used three-dimensional super-resolution fluorescence microscopy (interferometric photoactivated localization microscopy) to map nanoscale protein organization in focal adhesions. Our results reveal that integrins and actin are vertically separated by a \~{}40-nm focal adhesion core region consisting of multiple protein-specific strata: a membrane-apposed integrin signaling layer containing integrin cytoplasmic tails, focal adhesion kinase, and paxillin; an intermediate force-transduction layer containing talin and vinculin; and an uppermost actin-regulatory layer containing zyxin, vasodilator-stimulated phosphoprotein and α-actinin. By localizing amino- and carboxy-terminally tagged talins, we reveal talin’s polarized orientation, indicative of a role in organizing the focal adhesion strata. The composite multilaminar protein architecture provides a molecular blueprint for understanding focal adhesion functions.