Multiple Filopodia Research Articles

Qualitative platelet alterations in May — Hegglin anomaly

Introduction: May — Hegglin anomaly is an autosomal dominant mutation in the MYH9 gene that disrupts the dynamics of the cytoskeleton in megakaryocytes. Megakaryocyte dysfunction entails a violation of thrombocytopoiesis, which is manifested by macrothrombocytopenia, sometimes associated with bleeding. Aim: to study the structure and function of platelets in the family members with documented May — Hegglin anomaly. Patients and methods. The proband, her sister and their mother who all had a heterozygous mutation R1933X in the MYH9 gene were examined. The examination included complete blood count, peripheral blood microscopy, platelet flow cytometry, blood clot contraction kinetics, scanning and transmission electron microscopy of platelets. The control group included10 healthy donors. Results. In the proband and her sister the degree of contraction of blood clots was reduced. Unstimulated platelets displayed overexpression of P-selectin and active αIIbβ3 integrin. After TRAP-induced stimulation the proportion of platelets expressing P-selectin in the patients was below normal, indicating partial platelet refractoriness. Electron microscopy of the unstimulated platelets revealed macrothrombocytosis and multiple filopodia, as well as enlargement of the open canalicular system. Conclusion. The May — Hegglin anomaly caused by a heterozygous mutation R1933X in the MYH9 gene is accompanied by thrombocytopenia associated with structural and functional platelet defects. Partial platelet refractoriness and contractile dysfunction can impair the retraction of hemostatic clots, predisposing patients with MYH9-related disorders to bleeding.

A familial case of MYH9 gene mutation associated with multiple functional and structural platelet abnormalities

Mutations in the MYH9 gene result in macrothrombocytopenia often associated with hemorrhages. Here, we studied the function and structure of platelets in three family members with a heterozygous mutation R1933X in the MYH9 gene, characteristic of closely related disorders known as the May-Hegglin anomaly and Sebastian syndrome. The examination included complete blood count, blood smear microscopy, platelet flow cytometry (expression of P-selectin and active integrin αIIbβ3 before and after activation), the kinetics of platelet-driven contraction (retraction) of blood clots, as well as scanning/transmission electron microscopy of platelets. Despite severe thrombocytopenia ranging (36–86) × 109/l, none of the patients had hemorrhages at the time of examination, although they had a history of heavy menstruation, spontaneous ecchymosis, and postpartum hemorrhage. Flow cytometry showed background platelet activation, revealed by overexpression of P-selectin and active αIIbβ3 integrin above normal levels. After TRAP-induced stimulation, the fractions of platelets expressing P-selectin in the proband and her sister were below normal response, indicating partial platelet refractoriness. The initiation of clot contraction was delayed. Electron microscopy revealed giant platelets with multiple filopodia and fusion of α-granules with dilated open canalicular system, containing filamentous and vesicular inclusions. The novel concept implies that the R1933X mutation in the MYH9 gene is associated not only with thrombocytopenia, but also with qualitative structural and functional defects in platelets. Platelet dysfunction includes impaired contractility, which can disrupt the compaction of hemostatic clots, making the clots weak and permeable, therefore predisposing patients with MYH9 gene mutations to the hemorrhagic phenotype.

Construction of micro-grooved PCL/nanohydroxyapatite membranes by non-solvent induced phase separation method and its evaluation for use as a substrate for human periodontal ligament fibroblasts

Micro-patterned membrane constructs based on neat, and nanohydroxyapatite (HA)-blended poly(ε-caprolactone) were developed using the phase separation micro-molding (PSµM) technique. Physical, chemical, thermal, mechanical and in vitro biological properties of the constructs were evaluated. Human periodontal ligament fibroblasts (hPDLFs) were cultured on the surface of the patterned membranes. The effect of micro-patterning on the morphology and osteogenic differentiation capacity of cells was evaluated by SEM and AR-S staining. SEM findings revealed that hPDLFs were able to adhere to ridges, but generally tended to settle along microgroove pattern channels with multiple filopodia. Micro-groove structure led to a narrower elongation of the cytoplasm, orientation and directionality for migration along the groove. After 30 days of culture, the cells had grown to form multiple layers covering the entire patterned surface, while maintaining the alignment of the cells. Extracellular mineralization and osteogenic differentiation of hPDLFs was demonstrated by high AR-S staining, particularly on HA-blended constructs.

Isolation and characterization of an endoparasite from the culture of oleaginous microalga Graesiella sp. WBG-1

Oleaginous microalgae are an important potential feedstock for biodiesel production. However, a problem in mass cultivation of microalgae is the frequent occurrence of infection by algal parasites. In the present study, an endoparasite WZ01 was isolated from an open raceway pond of oleaginous Graesiella sp. WBG-1, and caused epidemics resulting in algal population collapse. According to 18S rDNA-based phylogenetic analyses, morphology, ultrastructure and life cycle, strain WZ01 was identified as a member of Aphelidea, Amoeboaphelidium protococcarum, although it had some minor differences from A. protococcarum X-5 and FD95. Examination via transmission electron microscopy demonstrated that zoospores of the parasite WZ01 were amoeboid, which can produce two types of pseudopodia (multiple filopodia and short anterior lamellipodium), but the pseudocilium was not observed. Interestingly, amoeboid zoospores contained numerous dense-body vesicles, which have not been previously described. Moreover, our results revealed the presence of a microtubule inside the penetration tube, indicating that the contents of the cyst were injected into the host not only by vacuole pressure but also by using a microtubule-mediated mechanism. In addition, of the 42 tested algal species, only cultures of coccoid green algae Chlorococcum sp. A213 and Chlorococcum sp. GP1 rapidly and intensively developed infections by parasite WZ01. This is the first report on endoparasitic infection in oleaginous coccoid green algae. Our results will enhance understanding of parasite–host relationships, which will be beneficial in developing strategies for infection control.

Bisphenol A Inhibits Cell Proliferation and Reduces the Motile Potential of Murine LM8 Osteosarcoma Cells.

The aim of this study was to examine the effect of bisphenol A (BPA) on the proliferation and motility potential of murine LM8 osteosarcoma cells. LM8 cells were treated for 3 days with or without 80 μM BPA. The effect of BPA on cell proliferation was determined by DNA measurement in the cultures and 5-bromo-2'-deoxyuridine (BrdU) incorporation study. Ethanol-fixed cells were stained with hematoxylin-eosin (H&E) to visualize cell morphology. Cell motility was assayed using inserts with uncoated membranes in invasion chambers. Expression of cell division cycle 42 (CDC42) was determined by immunofluorescence staining and western blotting. BPA reduced the DNA content of cultures and the number of BrdU-positive cells. BPA induced a change in morphology from cuboidal with multiple filopodia on the cell surface to spindle-shaped with a smooth cell surface. BPA-treated cells expressed less CDC42 and were less motile than untreated cells. BPA inhibited DNA replication and cell proliferation. BPA inhibited filopodia formation and motile potential by inhibiting CDC42 expression in LM8 cells.

A titanium surface with nano-ordered spikes and pores enhances human dermal fibroblastic extracellular matrix production and integration of collagen fibers

The acquisition of substantial dermal sealing determines the prognosis of percutaneous titanium-based medical devices or prostheses. A nano-topographic titanium surface with ordered nano-spikes and pores has been shown to induce periodontal-like connective tissue attachment and activate gingival fibroblastic functions. This in vitro study aimed to determine whether an alkali-heat (AH) treatment-created nano-topographic titanium surface could enhance human dermal fibroblastic functions and binding strength to the deposited collagen on the titanium surface.The surface topographies of commercially pure titanium machined discs exposed to two different AH treatments were evaluated. Human dermal fibroblastic cultures grown on the discs were evaluated in terms of cellular morphology, proliferation, extracellular matrix (ECM) and proinflammatory cytokine synthesis, and physicochemical binding strength of surface-deposited collagen.An isotropically-patterned, shaggy nano-topography with a sponge-like inner network and numerous well-organized, anisotropically-patterned fine nano-spikes and pores were observed on each nano-topographic surface type via scanning electron microscopy. In contrast to the typical spindle-shaped cells on the machined surfaces, the isotropically- and anisotropically-patterned nano-topographic titanium surfaces had small circular/angular cells containing contractile ring-like structures and elongated, multi-shaped cells with a developed cytoskeletal network and multiple filopodia and lamellipodia, respectively. These nano-topographic surfaces enhanced dermal-related ECM synthesis at both the protein and gene levels, without proinflammatory cytokine synthesis or reduced proliferative activity. Deposited collagen fibers were included in these surfaces and sufficiently bound to the nano-topographies to resist the physical, enzymatic and chemical detachment treatments, in contrast to machined surfaces.Well-organized, isotropically-/anisotropically-patterned, nano-topographic titanium surfaces with AH treatment-created nano-spikes and pores enhanced human dermal fibroblastic ECM synthesis and established sufficient mechanical integration between the surfaces and ECM to resist various detachment treatments used to experimentally mimic overloading and inflammation.

Human aortic endothelial cell morphology influenced by topography of porous silicon substrates.

Porous silicon has received much attention because of its optical properties and for its usefulness in cell-based biosensing, drug delivery, and tissue engineering applications. Surface properties of the biomaterial are associated with cell adhesion and with proliferation, migration, and differentiation. The present article analyzes the behavior of human aortic endothelial cells in macro- and nanoporous collagen-modified porous silicon samples. On both substrates, cells are well adhered and numerous. Confocal microscopy and scanning electron microscopy were employed to study the effects of porosity on the morphology of the cells. On macroporous silicon, filopodia is not observed but the cell spreads on the surface, increasing the lamellipodia surface which penetrates the macropore. On nanoporous silicon, multiple filopodia were found to branch out from the cell body. These results demonstrate that the pore size plays a key role in controlling the morphology and growth rate of human aortic endothelial cells, and that these forms of silicon can be used to control cell development in tissue engineering as well as in basic cell biology research.

Promoting filopodial elongation in neurons by membrane-bound magnetic nanoparticles

Filopodia are 5-10μm long processes that elongate by actin polymerization, and promote axon growth and guidance by exerting mechanical tension and by molecular signaling. Although axons elongate in response to mechanical tension, the structural and functional effects of tension specifically applied to growth cone filopodia are unknown. Here we developed a strategy to apply tension specifically to retinal ganglion cell (RGC) growth cone filopodia through surface-functionalized, membrane-targeted superparamagnetic iron oxide nanoparticles (SPIONs). When magnetic fields were applied to surface-bound SPIONs, RGC filopodia elongated directionally, contained polymerized actin filaments, and generated retrograde forces, behaving as bona fide filopodia. Data presented here support the premise that mechanical tension induces filopodia growth but counter the hypothesis that filopodial tension directly promotes growth cone advance. Future applications of these approaches may be used to induce sustained forces on multiple filopodia or other subcellular microstructures to study axon growth or cell migration. From the Clinical EditorMechanical tension to the tip of filopodia is known to promote axonal growth. In this article, the authors used superparamagnetic iron oxide nanoparticles (SPIONs) targeted specifically to membrane molecules, then applied external magnetic field to elicit filopodial elongation, which provided a tool to study the role of mechanical forces in filopodia dynamics and function.

Single-neuron tracing study of thalamocortical projections arising from the rat ventral medial nucleus by using viral vectors

Filopodia are 5-10 μm long processes that elongate by actin polymerization, and promote axon growth and guidance by exerting mechanical tension and by molecular signaling. Although axons elongate in response to mechanical tension, the structural and functional effects of tension specifically applied to growth cone filopodia are unknown. Here we developed a strategy to apply tension specifically to retinal ganglion cell (RGC) growth cone filopodia through surface-functionalized, membrane-targeted superparamagnetic iron oxide nanoparticles (SPIONs). When magnetic fields were applied to surface-bound SPIONs, RGC filopodia elongated directionally, contained polymerized actin filaments, and generated retrograde forces, behaving as bona fide filopodia. Data presented here support the premise that mechanical tension induces filopodia growth but counter the hypothesis that filopodial tension directly promotes growth cone advance. Future applications of these approaches may be used to induce sustained forces on multiple filopodia or other subcellular microstructures to study axon growth or cell migration.Mechanical tension to the tip of filopodia is known to promote axonal growth. In this article, the authors used superparamagnetic iron oxide nanoparticles (SPIONs) targeted specifically to membrane molecules, then applied external magnetic field to elicit filopodial elongation, which provided a tool to study the role of mechanical forces in filopodia dynamics and function.

Abstract 5267: Mammary stromal fibroblast activity is modulated by HoxC6: impacts on growth and metastatic potential

Abstract Crosstalk between mammary epithelium and stromal cell types, including fibroblasts and adipocytes is 1) essential for mammary gland development 2) likely impacts breast cancer potential and 3) significantly impacts the growth, invasion and metastasis of tumor cells. Although well recognized, molecular mechanisms important in stromal-epithelial crosstalk are still poorly understood. The purpose of this study is to better elucidate one potentially significant mechanism involving stromal fibroblast to epithelial paracrine signaling in the mammary gland. We previously demonstrated that HoxC6 was required for mammary epithelial cell growth and ductal branching morphogenesis using mouse models and 3-dimensional in vitro culture of human mammary epithelial cell lines. We also demonstrated that constitutive ErbB2/Her2/Neu receptor activation in epithelium of transgenics is sufficient to rescue HoxC6 deficiency in epithelial growth but not branching. Significantly, this model also shows that HoxC6 null stroma facilitates tumor metastasis. Here, we focus attention on stromal fibroblasts (vimentin positive cells) from wild type and HoxC6 null prepubertal mouse thoracic mammary glands (TMGs). When cultured in vitro, HoxC6 null TMG fibroblasts display multiple filopodia and extended cellular processes that could indicate an activated myofibroblast phenotype. In contrast, cultured wild type TMG fibroblasts appear quite similar to mouse embryonic fibroblasts. Quantitative comparisons of gene expression patterns in isolated fibroblasts indicate that HoxC6 null fibroblasts are more activated than wild type fibroblasts. Activation markers studied include alpha smooth muscle actin (ACTA2/SMA), S100A4 (Fsp1) and Wnt1-induced secreted protein 1 (Wisp1). Since estrogens are know to repress HoxC6 expression and are implicated in modulating paracrine stromal-to-epithelial interactions, stromal fibroblasts were isolated and cultured under estrogen-free conditions and treated with 17beta-estradiol, bisphenol A and vitamin D. In addition, stromal fibroblasts were co-cultured with mammary epithelial stem cells and stem-like mammary tumor cells to study the impact of stromal fibroblast HoxC6 status on epithelial cell activities. The results obtained will be discussed as they pertain to stromal-to-epithelial signaling in mammary gland development and in the growth, invasion and metastasis of tumor cells. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5267.

Actin-Rich Protrusions and Nonlocalized GTPase Activation in Merlin-Deficient Schwannomas

Schwannomas lack both alleles for the tumor suppressor Merlin, a cytoskeleton-membrane linker. Previous results showed increased cell spreading of schwannoma cells, but little is known about the underlying mechanisms. Electron microscopy reveals that schwannoma cells not only show more lamellipodia/ruffles but also multiple filopodia. We show that Cdc42, important in filopodia formation, is activated. Both Rac1 and Cdc42 are found all around the cell periphery and in colocalization with their effector phospho-p21 activated kinase in human schwannoma cells. We therefore claim that Rac1 and Cdc42 are activated in a nonlocalized manner, which explains the disperse distribution of lamellipodia/ruffles and filopodia. Using live cell imaging, we further demonstrate continuous remodeling of the many actin-rich protrusions in schwannoma cells. The underlying cytoskeleton of these structures is thin and extensively branched. The actin-related protein 2/3 complex, a major regulator of actin branching, is enriched in the many lamellipodia and ruffles of human primary schwannoma cells. We suggest that the Merlin deficiency in human primary schwannoma cells leads to a random, nonlocalized activation of Rac1 and Cdc42, inducing many actin-rich protrusion zones, not only at the leading edge but also all around the cell periphery. Their nondirectional occurrence together with the continuous and highly dynamic actin remodeling results in the dedifferentiation of these tumor cells.

Pioneer growth cone steering along a series of neuronal and non- neuronal cues of different affinities

We have analyzed the morphology of over 5000 Ti1 pioneer growth cones labeled with anti-HRP, which reveals the disposition of axons, growth cone branches, and filopodia. Ti1 axon pathways typically consist of a sequence of 7 characteristically oriented segments, with a single, distinct reorientation point between each segment. Growth cones exhibit the same orientations and reorientations in a given region as do axon segments at later stages. The single, distinct reorientations suggest that growth cones make discrete switches between guidance cues as they grow. Ti1 growth cones are guided by various types of cues. A set of 3 immature identified neurons serves as nonadjacent guidepost cells and lies at the proximal end of 3 of the axon segments. To form another segment, growth cones reorient along a limb segment boundary within the epithelium. Growth cones also respond consistently to, and orient toward, a specific mesodermal cell, which may be a muscle pioneer. Thus, growth cones respond to at least 3 different types of cells in the leg. Ti1 growth cones exhibit a hierarchy of affinity for these cues. Guidepost neurons are the dominant cues in that contact with them reorients growth cones from guidance by the other types of cues. Growth cone branches are exclusively oriented to specific cues. Growth cones reorient by extending a branch directly to the cue of highest affinity and by withdrawing any branches that are extended to a cue of lesser affinity. A single filopodium in direct contact with a guidepost neuron can reorient a growth cone that still has multiple filopodia or even prominent branches specifically oriented to a previous cue of lesser affinity. These observations suggest that growth cone steering may not result simply from passive adhesion and filopodial traction, but may involve more active processes.