RESUMEN
Filopodia, dynamic membrane protrusions driven by polymerization of an actin filament core, can adhere to the extracellular matrix and experience both external and cell-generated pulling forces. The role of such forces in filopodia adhesion is however insufficiently understood. Here, we study filopodia induced by overexpression of myosin X, typical for cancer cells. The lifetime of such filopodia positively correlates with the presence of myosin IIA filaments at the filopodia bases. Application of pulling forces to the filopodia tips through attached fibronectin-coated laser-trapped beads results in sustained growth of the filopodia. Pharmacological inhibition or knockdown of myosin IIA abolishes the filopodia adhesion to the beads. Formin inhibitor SMIFH2, which causes detachment of actin filaments from formin molecules, produces similar effect. Thus, centripetal force generated by myosin IIA filaments at the base of filopodium and transmitted to the tip through actin core in a formin-dependent fashion is required for filopodia adhesion.
Asunto(s)
Forminas/metabolismo , Miosinas/metabolismo , Neoplasias/metabolismo , Miosina Tipo IIA no Muscular/metabolismo , Seudópodos/fisiología , Citoesqueleto de Actina , Animales , Células COS , Chlorocebus aethiops , Forminas/antagonistas & inhibidores , Forminas/genética , Forminas/ultraestructura , Regulación Neoplásica de la Expresión Génica , Células HeLa , Humanos , Proteínas de Microfilamentos , Miosina Tipo IIA no Muscular/antagonistas & inhibidores , Miosina Tipo IIA no Muscular/genética , Miosina Tipo IIA no Muscular/ultraestructura , Seudópodos/patología , Tionas/farmacología , Uracilo/análogos & derivados , Uracilo/farmacologíaRESUMEN
The quality of super resolution images obtained by stochastic single-molecule microscopy critically depends on image analysis algorithms. We find that the choice of background estimator is often the most important determinant of reconstruction quality. A variety of techniques have found use, but many have a very narrow range of applicability depending upon the characteristics of the raw data. Importantly, we observe that when using otherwise accurate algorithms, unaccounted background components can give rise to biases on scales defeating the purpose of super-resolution microscopy. We find that a temporal median filter in particular provides a simple yet effective solution to the problem of background estimation, which we demonstrate over a range of imaging modalities and different reconstruction methods.
Asunto(s)
Aumento de la Imagen/métodos , Interpretación de Imagen Asistida por Computador/métodos , Microscopía Nuclear/métodos , Actinas/ultraestructura , Algoritmos , Carbocianinas , Línea Celular Tumoral , Colorantes Fluorescentes , Células HeLa , Humanos , Miosina Tipo IIA no Muscular/ultraestructura , Vinculina/ultraestructuraRESUMEN
This study describes a novel cytoskeletal array in fiber cells of the ocular lens of the rat and shows its relationship to the classical terminal web of other epithelial tissues. Naive adult Sprague-Dawley rats (n = 28) were utilized. F-actin, fodrin, myosin IIA, and CP49 distribution was assessed in anterior and posterior polar sections. For functional analysis, lenses were cultured with or without cytochalasin-D for 3 hr, then processed for confocal microscopy or assessed by laser scan analysis along sutures. Phalloidin labeling demonstrated a dense mesh of F-actin adjacent to posterior sutural domains to a subcapsular depth of 400 µm. Anterior polar sections revealed a comparable actin structure adjacent to anterior suture branches however, it was not developed in superficial fibers. Fodrin and myosin were localized within the web-like actin apparatus. The data was used to construct a model showing that the cytoskeletal array is located within the blunt, variable-width fiber ends that abut at sutures such that the "terminal web" flanks the suture on either side. Treatment with cytochalasin-D resulted in partial disassembly of the "terminal web" and perturbed cellular organization. Laser scan analysis revealed that cytochalasin-D treated lenses had significantly greater focal variability than control lenses (P = 0.020). We conclude that cortical fibers of rat lenses contain a bipolar structure that is structurally and compositionally analogous to classical terminal webs. The results indicate that the lens "terminal web" functions to stabilize lens fiber ends at sutures thus minimizing structural disorder, which in turn, promotes the establishment and maintenance of lens transparency.
Asunto(s)
Citoesqueleto/fisiología , Citoesqueleto/ultraestructura , Cristalino/citología , Cristalino/fisiología , Actinas/análisis , Actinas/fisiología , Actinas/ultraestructura , Animales , Proteínas Portadoras/análisis , Proteínas Portadoras/fisiología , Proteínas Portadoras/ultraestructura , Citocalasina D/farmacología , Citoesqueleto/efectos de los fármacos , Proteínas del Ojo/análisis , Proteínas del Ojo/fisiología , Proteínas del Ojo/ultraestructura , Proteínas de Filamentos Intermediarios/análisis , Proteínas de Filamentos Intermediarios/fisiología , Proteínas de Filamentos Intermediarios/ultraestructura , Cristalino/química , Proteínas de Microfilamentos/análisis , Proteínas de Microfilamentos/fisiología , Proteínas de Microfilamentos/ultraestructura , Microscopía Confocal , Miosina Tipo IIA no Muscular/análisis , Miosina Tipo IIA no Muscular/fisiología , Miosina Tipo IIA no Muscular/ultraestructura , Inhibidores de la Síntesis del Ácido Nucleico/farmacología , Ratas , Ratas Sprague-DawleyRESUMEN
Loads on molecular motors regulate and coordinate their function. In a study that directly measures properties of internally strained myosin 2 heads bound to actin, we find that human nonmuscle myosins 2A and 2B show marked load-dependent changes in kinetics of ADP release but not in nucleotide binding. We show that the ADP release rate constant is increased 4-fold by the assisting load on one head and decreased 5-fold (for 2A) or 12-fold (for 2B) by the resisting load on the other. Thus these myosins, especially 2B, have marked mechanosensitivity of product release. By regulating the actin attachment of myosin heads, this provides a basis for energy-efficient tension maintenance without obstructing cellular contractility driven by other motors such as smooth muscle myosin. Whereas forward load accelerates the cycle of interaction with actin, resistive load increases duty ratio to favor tension maintenance by two-headed attachment.
Asunto(s)
Miosina Tipo IIA no Muscular/química , Miosina Tipo IIA no Muscular/metabolismo , Miosina Tipo IIB no Muscular/química , Miosina Tipo IIB no Muscular/metabolismo , Actinas/metabolismo , Adenosina Difosfato/metabolismo , Adenosina Trifosfato/metabolismo , Fenómenos Biomecánicos , Humanos , Cinética , Modelos Biológicos , Proteínas Motoras Moleculares/química , Proteínas Motoras Moleculares/metabolismo , Subfragmentos de Miosina/metabolismo , Subfragmentos de Miosina/ultraestructura , Miosina Tipo IIA no Muscular/ultraestructura , Miosina Tipo IIB no Muscular/ultraestructura , Unión Proteica , TermodinámicaRESUMEN
Nonmuscle myosin II is among the most abundant forms of myosin in nerve growth cones. At least two isoforms of myosin II (A and B) that have overlapping but distinct distributions are found in growth cones. It appears that both myosin IIA and IIB may be necessary for normal nerve outgrowth and motility, but the molecular interactions responsible for their activity remain unclear. For instance, it is unknown if these myosin II isoforms produce bipolar "minifilaments" in growth cones similar to those observed in other nonmuscle cells. To determine if minifilaments are present in growth cones, we modified the electron microscopy preparative procedures used to detect minifilaments in other cell types. We found structures that appeared very similar to bipolar minifilaments found in noneuronal cells. They also labeled with antibodies to either myosin IIA or IIB. Thus, the activity of myosin II in growth cones is likely to be similar to that in other nonmuscle cells. Bipolar filaments interacting with oppositely oriented actin filaments will produce localized contractions or exert tension on actin networks. This activity will be responsible for the myosin II dependent motility in growth cones.