Despite recent advances in our understanding of the components and spatial regulation contractile ring (CR), precise ultrastructure actin myosin II within animal cell CR remains an unanswered question. We used superresolution light microscopy platinum replica transmission electron (TEM) to determine structural organization isolated cortical cytoskeletons prepared from dividing sea urchin embryos. Three-dimensional structured illumination indicated that CR, filaments were organized into...

Recent studies have investigated the dendritic actin cytoskeleton of cell edge's lamellipodial (LP) region by experimentally decreasing activity filament nucleator and branch former, Arp2/3 complex. Here we extend these via pharmacological inhibition complex in sea urchin coelomocytes, cells that possess an unusually broad LP display correspondingly exaggerated centripetal flow. Using light electron microscopy, demonstrate drug CK666 dramatically altered architecture, slowed flow, drove a...

Sea urchin coelomocytes represent an excellent experimental model system for studying retrograde flow. Their extreme flatness allows microscopic visualization. discoid shape provides a radially symmetric geometry, which simplifies analysis of the flow pattern. Finally, nonmotile nature cells to be analyzed in absence cell translocation. In this study we have begun mechanism by characterizing its kinetic and structural properties. The supramolecular organization actin myosin II was...

Kinesin was previously immunolocalized to mitotic apparatuses (MAs) of early sea urchin blastomeres (Scholey, J.M., M.E. Porter, P.M. Grissom, and J.R. McIntosh. 1985. Nature [Lond.]. 318:483-486). Here we report evidence that this MA-associated motor protein is a conventional membrane-bound kinesin, rather than kinesin-like protein. Our includes the observation deduced amino acid sequence kinesin heavy chain characteristic kinesin. In addition, immunolocalizations using antibodies...

Using an antiserum produced against a purified calsequestrin-like (CSL) protein from microsomal fraction of sea urchin eggs, we performed light and electron microscopic immunocytochemical localizations on eggs embryos in the first cell cycle. The CSL has been found to bind Ca++ similarly calsequestrin, well-characterized storage sarcoplasmic reticulum muscle cells. In semi-thin frozen sections unfertilized immunofluorescent staining revealed tubuloreticular network throughout cytoplasm....

The current paradigm proposes that the innate immune systems of invertebrates are much more complex than previously thought. highly diverse 185/333 gene family in purple sea urchin encodes a closely related proteins varying length and sequence composition. Subsets small phagocytes polygonal cells express with localization on surface within perinuclear vesicles both cell types. In short-term cultures, coelomocytes form aggregates progress to syncytia thought be equivalent encapsulation vivo....

We have recently used domain-specific monoclonal antibodies (mAbs) to immunofluorescently localize kinesin vesicle-like structures in the cytoplasm of sea urchin coelomocytes. In order characterize further these localization patterns we examined distribution with respect arrangement microtubules (MTs) and various organelles. double-label experiments involving immunofluorescent staining (using a mixture mAbs SUK2, 4 5), MTs were labeled an antiserum against tubulin, endoplasmic reticulum (ER)...

In the sea urchin embryo, gastrulation is characterized by ingression and directed cell migration of primary mesenchyme cells (PMCs), as well invagination convergent extension endomesoderm. Like all shape changes, individual collective motility orchestrated Rho family GTPases their modulation actomyosin cytoskeleton. And while endomesoderm specification has been intensively studied in echinoids, much less known about proximate regulators driving motility. Toward these ends, we employed...

We have utilized immunoblotting and light microscopic immunofluorescent staining methods to examine the expression localization of sea urchin kinesin-II, a heterotrimeric plus end-directed microtubule motor protein (previously referred as KRP(85/95)), in sand dollar sperm. demonstrate presence 85 K 115 subunits kinesin-II sperm localize these proteins flagella midpiece. The pattern is punctate discontinuous, it quite distinct from continuous labeling present labeled with anti-flagellar...

The dogfish shark (Squalus acanthias) rectal gland (SRG) cell has served as a model experimental system for investigating the relationship between actin cytoskeleton and volume regulation. Previous reports employing conventional fluorescence microscopy of tissue slices have shown that cells exposed to high external K+ hypotonically-induced swelling displayed fading F-actin staining intensity, particularly at basolateral borders. However, spectroscopic measurement present in similarly treated...

The actomyosin purse string is an evolutionarily conserved contractile structure that involved in cytokinesis, morphogenesis, and wound healing. Recent studies suggested crucial for the closure of wounds single cells. In present study, morphological pharmacological methods were used to investigate role this peripheral cytoplasm sea urchin coelomocytes. These discoidal shaped cells underwent a dramatic form actin-based centripetal/retrograde flow occasionally opened closed spontaneous their...

In motile cells, the activities of different Rho family GTPases are spatially segregated within cell, and during cytokinesis there is evidence that this may also be case. But while Rho's role as central organizer for contractile ring assembly well established, Rac branched actin networks it promotes less understood. To characterize contributions these proteins cytokinesis, we manipulated Arp2/3 activity mitosis meiosis in sea urchin embryos star oocytes. While neither nor were essential...

Abstract Previous studies have suggested that the actin‐based centripetal flow process in sea urchin coelomocytes is result of a two‐part mechanism, actin polymerization at cell edge coupled with actomyosin contraction center. In present study, we extended testing this model by attempting to stimulate via treatment phosphatase inhibitor Calyculin A (CalyA). The effects drug were studied using digitally‐enhanced video microscopy living cells combined immunofluorescent localization and...

Marine elasmobranch rectal gland is a specialized, osmoregulatory organ composed of numerous blind-ended, branched tubules emptying into central duct. To date, NaCl excretion has been its only described function. Here we use isolated tubule fragments from dogfish shark ( Squalus acanthias), fluorescent xenobiotics, and confocal microscopy to describe second function, xenobiotic excretion. Isolated rapidly transported the organic anion sulforhodamine 101 bath lumen. Luminal accumulation was...

Multidrug resistance-associated proteins 1 and 2 (Mrp1 Mrp2) are thought to mediate low-affinity ATP-dependent transport of reduced glutathione (GSH), but there is as yet no direct evidence for this hypothesis. The present study examined whether livers from the little skate ( Raja erinacea) express an Mrp2 homologue liver membrane vesicles exhibit GSH activity. Antibodies directed against mammalian Mrp2-specific epitopes labeled a 180-kDa protein band in plasma membranes stained canaliculi...