RESUMEN
Neural stem and progenitor cells (NSPCs) are heterogeneous populations of self-renewing stem cells and more committed progenitors that differentiate into neurons, astrocytes, and oligodendrocytes. Accurately identifying and characterizing the different progenitor cells in this lineage has continued to be a challenge for the field. We found previously that populations of NSPCs with more neurogenic progenitors (NPs) can be distinguished from those with more astrogenic progenitors (APs) by their inherent biophysical properties, specifically the electrophysiological property of whole cell membrane capacitance, which we characterized with dielectrophoresis (DEP). Here, we hypothesize that inherent electrophysiological properties are sufficient to define NPs and APs and test this by determining whether isolation of cells solely by these properties specifically separates NPs and APs. We found NPs and APs are enriched in distinct fractions after separation by electrophysiological properties using DEP. A single round of DEP isolation provided greater NP enrichment than sorting with PSA-NCAM, which is considered an NP marker. Additionally, cell surface N-linked glycosylation was found to significantly affect cell fate-specific electrophysiological properties, providing a molecular basis for the cell membrane characteristics. Inherent plasma membrane biophysical properties are thus sufficient to define progenitor cells of differing fate potential in the neural lineage, can be used to specifically isolate these cells, and are linked to patterns of glycosylation on the cell surface.
Asunto(s)
Astrocitos/citología , Fenómenos Biofísicos , Linaje de la Célula , Membrana Celular/fisiología , Células-Madre Neurales/citología , Neuronas/citología , Animales , Separación Celular , Tamaño de la Célula , Fenómenos Electrofisiológicos , Glicosilación , Potenciales de la Membrana , Ratones , MicrofluídicaRESUMEN
The emergence of processes from cells often involves interactions between microtubules and microfilaments. Interactions between these two cytoskeletal systems are particularly apparent in neuronal growth cones. The juvenile isoform of the neuronal microtubule-associated protein 2 (MAP2c) is present in growth cones, where we hypothesize it mediates interactions between microfilaments and microtubules. To approach this problem in vivo, we used the human melanoma cell, M2, which lacks actin-binding protein-280 (ABP-280) and forms membrane blebs, which are not seen in wild-type or ABP-transfected cells. The microinjection of tau or mature MAP2 rescued the blebbing phenotype; MAP2c not only caused cessation of blebbing but also induced the formation of two distinct cellular structures. These were actin-rich lamellae, which often included membrane ruffles, and microtubule-bearing processes. The lamellae collapsed after treatment with cytochalasin D, and the processes retracted after treatment with colchicine. MAP2c was immunocytochemically visualized in zones of the cell that were devoid of tubulin, such as regions within the lamellae and in association with membrane ruffles. In vitro rheometry confirmed that MAP2c is an efficient actin gelation protein capable of organizing actin filaments into an isotropic array at very low concentrations; tau and mature MAP2 do not share this rheologic property. These results suggest that MAP2c engages in functionally specific interactions not only with microtubules but also with microfilaments.
Asunto(s)
Citoesqueleto de Actina/fisiología , Actinas/metabolismo , Proteínas Portadoras/metabolismo , Melanoma/metabolismo , Proteínas de Microfilamentos/metabolismo , Proteínas Asociadas a Microtúbulos/fisiología , Microtúbulos/fisiología , Citoesqueleto de Actina/efectos de los fármacos , Actinas/efectos de los fármacos , Proteínas Portadoras/efectos de los fármacos , Tamaño de la Célula/efectos de los fármacos , Tamaño de la Célula/fisiología , Humanos , Melanoma/patología , Proteínas de Microfilamentos/efectos de los fármacos , Microinyecciones , Microtúbulos/efectos de los fármacos , Células Tumorales Cultivadas , Proteínas tau/farmacologíaRESUMEN
The Arp2/3 complex and filamin A (FLNa) branch actin filaments. To define the role of these actin-binding proteins in cellular actin architecture, we compared the morphology of FLNa-deficient human melanoma (M2) cells and three stable derivatives of these cells expressing normal FLNa concentrations. All the cell lines contain similar amounts of the Arp2/3 complex. Serum addition causes serum-starved M2 cells to extend flat protrusions transiently; thereafter, the protrusions turn into spherical blebs and the cells do not crawl. The short-lived lamellae of M2 cells contain a dense mat of long actin filaments in contrast to a more three-dimensional orthogonal network of shorter actin filaments in lamellae of identically treated FLNa-expressing cells capable of translational locomotion. FLNa-specific antibodies localize throughout the leading lamellae of these cells at junctions between orthogonally intersecting actin filaments. Arp2/3 complex-specific antibodies stain diffusely and label a few, although not the same, actin filament overlap sites as FLNa antibody. We conclude that FLNa is essential in cells that express it for stabilizing orthogonal actin networks suitable for locomotion. Contrary to some proposals, Arp2/3 complex-mediated branching of actin alone is insufficient for establishing an orthogonal actin organization or maintaining mechanical stability at the leading edge.
Asunto(s)
Citoesqueleto de Actina/fisiología , Actinas/metabolismo , Proteínas Contráctiles/metabolismo , Proteínas del Citoesqueleto , Proteínas de Microfilamentos/metabolismo , Citoesqueleto de Actina/metabolismo , Citoesqueleto de Actina/ultraestructura , Proteína 2 Relacionada con la Actina , Proteína 3 Relacionada con la Actina , Movimiento Celular/fisiología , Filaminas , Técnica del Anticuerpo Fluorescente Indirecta , Humanos , Melanoma , Microscopía Electrónica/métodos , Células Tumorales CultivadasRESUMEN
Doublecortin (DCX) is required for normal migration of neurons into the cerebral cortex, since mutations in the human gene cause a disruption of cortical neuronal migration. To date, little is known about the distribution of DCX protein or its function. Here, we demonstrate that DCX is expressed in migrating neurons throughout the central and peripheral nervous system during embryonic and postnatal development. DCX protein localization overlaps with microtubules in cultured primary cortical neurons, and this overlapping expression is disrupted by microtubule depolymerization. DCX coassembles with brain microtubules, and recombinant DCX stimulates the polymerization of purified tubulin. Finally, overexpression of DCX in heterologous cells leads to a dramatic microtubule phenotype that is resistant to depolymerization. Therefore, DCX likely directs neuronal migration by regulating the organization and stability of microtubules.
Asunto(s)
Movimiento Celular/fisiología , Proteínas Asociadas a Microtúbulos/biosíntesis , Neuronas/metabolismo , Neuropéptidos/biosíntesis , Fosfoproteínas/biosíntesis , Animales , Animales Recién Nacidos , Western Blotting , Movimiento Celular/efectos de los fármacos , Células Cultivadas , Corteza Cerebral/citología , Corteza Cerebral/crecimiento & desarrollo , Corteza Cerebral/metabolismo , Colchicina/farmacología , Proteínas de Dominio Doblecortina , Proteína Doblecortina , Humanos , Inmunohistoquímica , Hibridación in Situ , Ratones , Microtúbulos/efectos de los fármacos , Microtúbulos/metabolismo , Microtúbulos/ultraestructura , Mitosis , Neuronas/efectos de los fármacos , Neuronas/ultraestructura , Células de Purkinje/citología , Células de Purkinje/metabolismo , ARN Mensajero/biosíntesis , Ratas , Ratas Long-Evans , Tubulina (Proteína)/aislamiento & purificación , Tubulina (Proteína)/metabolismoRESUMEN
LIS1, a microtubule-associated protein, is required for neuronal migration, but the precise mechanism of LIS1 function is unknown. We identified a LIS1 interacting protein encoded by a mouse homolog of NUDE, a nuclear distribution gene in A. nidulans and a multicopy suppressor of the LIS1 homolog, NUDF. mNudE is located in the centrosome or microtubule organizing center (MTOC), and interacts with six different centrosomal proteins. Overexpression of mNudE dissociates gamma-tubulin from the centrosome and disrupts microtubule organization. Missense mutations that disrupt LIS1 function block LIS1-mNudE binding. Moreover, misexpression of the LIS1 binding domain of mNudE in Xenopus embryos disrupts the architecture and lamination of the CNS. Thus, LIS1-mNudE interactions may regulate neuronal migration through dynamic reorganization of the MTOC.
Asunto(s)
Sistema Nervioso Central/embriología , Sistema Nervioso Central/metabolismo , Centrosoma/metabolismo , Proteínas Fúngicas/metabolismo , Proteínas Asociadas a Microtúbulos/metabolismo , 1-Alquil-2-acetilglicerofosfocolina Esterasa , Animales , Células COS , Embrión no Mamífero/anomalías , Embrión no Mamífero/patología , Proteínas Fúngicas/genética , Regulación del Desarrollo de la Expresión Génica , Ratones , Proteínas Asociadas a Microtúbulos/genética , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Unión Proteica/genética , Estructura Terciaria de Proteína/genética , ARN Mensajero/biosíntesis , Homología de Secuencia de Aminoácido , Especificidad por Sustrato/fisiología , Tubulina (Proteína)/metabolismo , Técnicas del Sistema de Dos Híbridos , XenopusRESUMEN
Neuronal cytoskeletal elements such as neurofilaments, F-actin, and microtubules are actively translocated by an as yet unidentified mechanism. This report describes a novel interaction between neurofilaments and microtubule motor proteins that mediates the translocation of neurofilaments along microtubules in vitro. Native neurofilaments purified from spinal cord are transported along microtubules at rates of 100-1000 nm/s to both plus and minus ends. This motion requires ATP and is partially inhibited by vanadate, consistent with the activity of neurofilament-bound molecular motors. Motility is in part mediated by the dynein/dynactin motor complex and several kinesin-like proteins. This reconstituted motile system suggests how slow net movement of cytoskeletal polymers may be achieved by alternating activities of fast microtubule motors.
Asunto(s)
Dineínas/fisiología , Filamentos Intermedios/fisiología , Proteínas Asociadas a Microtúbulos/fisiología , Microtúbulos/fisiología , Neuronas/fisiología , Animales , Bovinos , Complejo Dinactina , Dineínas/aislamiento & purificación , Filamentos Intermedios/ultraestructura , Proteínas Asociadas a Microtúbulos/aislamiento & purificación , Microtúbulos/ultraestructura , Movimiento , Neuronas/ultraestructura , Ratas , Médula Espinal/citologíaRESUMEN
The recent demonstration that the fast axonal transport motors kinesin and dynein participate in axonal transport of neurofilaments--known to undergo slow transport--supports and extends recent studies indicating that some neurofilaments exhibit alternating bursts of fast axonal transport interspersed with periods of non-motility. In addition, these findings unify both certain aspects of axonal transport and neurofilament biology. We discuss these data herein in the context of both older and more recent studies of neurofilament dynamics.
Asunto(s)
Dineínas/fisiología , Cinesinas/fisiología , Proteínas de Neurofilamentos/metabolismo , Animales , Transporte Biológico/fisiología , FosforilaciónRESUMEN
Triple-negative breast cancer (TNBC) is a highly aggressive and metastatic form of breast cancer that lacks the estrogen, progesterone and HER2 receptors and is resistant to targeted and hormone therapies. TNBCs express high levels of the transmembrane glycoprotein, complement C1r/C1s, Uegf, Bmp1 (CUB)-domain containing protein 1 (CDCP1), which has been correlated with the aggressiveness and poor prognosis of multiple carcinomas. Full-length CDCP1 (flCDCP1) can be proteolytically cleaved, resulting in a cleaved membrane-bound isoform (cCDCP1). CDCP1 is phosphorylated by Src family kinases in its full-length and cleaved states, which is important for its pro-metastatic signaling. We observed that cCDCP1, compared with flCDCP1, induced a dramatic increase in phosphorylation of the migration-associated proteins: PKCδ, ERK1/2 and p38 mitogen-activated protein kinase in HEK 293T. In addition, only cCDCP1 induced migration of HEK 293T cells and rescued migration of the TNBC cell lines expressing short hairpin RNA against CDCP1. Importantly, we found that only cCDCP1 is capable of dimerization, which can be blocked by expression of the extracellular portion of cCDCP1 (ECC), indicating that dimerization occurs through CDCP1's ectodomain. We found that ECC inhibited phosphorylation of PKCδ and migration of TNBC cells in two-dimensional culture. Furthermore, ECC decreased cell invasiveness, inhibited proliferation and stimulated apoptosis of TNBC cells in three-dimensional culture, indicating that the cCDCP1 dimer is an important contributor to TNBC aggressiveness. These studies have important implications for the development of a therapeutic to block CDCP1 activity and TNBC metastasis.
Asunto(s)
Antígenos CD/genética , Moléculas de Adhesión Celular/genética , Proteínas de Neoplasias/genética , Isoformas de Proteínas/genética , Neoplasias de la Mama Triple Negativas/genética , Antígenos CD/química , Antígenos de Neoplasias , Apoptosis , Adhesión Celular/genética , Moléculas de Adhesión Celular/química , Movimiento Celular/genética , Dimerización , Células HEK293 , Humanos , Proteínas de Neoplasias/biosíntesis , Proteínas de Neoplasias/química , Fosforilación , Isoformas de Proteínas/biosíntesis , Transducción de Señal/genética , Neoplasias de la Mama Triple Negativas/patología , Proteínas Quinasas p38 Activadas por Mitógenos/genéticaRESUMEN
Cell movement and resistance to mechanical forces are largely governed by the cytoskeleton, a three-dimensional network of protein filaments that form viscoelastic networks within the cytoplasm. The cytoskeleton underlying the plasma membrane of most cells is rich in actin filaments whose assembly and disassembly are regulated by actin binding proteins that are stimulated or inhibited by signals received and transmitted at the membrane/cytoplasm interface. Inositol phospholipids, or phosphoinositides, are potent regulators of many actin binding proteins, and changes in the phosphorylation of specific phosphoinositide species or in their spatial localization are associated with cytoskeletal remodeling in vitro. This review will focus on recent studies directed at defining the structural features of phosphoinositide binding sites in actin binding proteins and on the influence of the physical state of phosphoinositides on their ability to interact with their target proteins.
Asunto(s)
Proteínas del Citoesqueleto/metabolismo , Citoesqueleto/química , Metabolismo de los Lípidos , Fosfatidilinositoles/metabolismo , Secuencia de Aminoácidos , Animales , Citoesqueleto/metabolismo , Humanos , Lípidos/química , Datos de Secuencia Molecular , Estructura Molecular , Unión ProteicaRESUMEN
Phosphatidylinositol bisphosphate (PIP2) serves as a precursor for diacylglycerol and inositol trisphosphate in signal transduction cascades and regulates the activities of several actin binding proteins that influence the organization of the actin cytoskeleton. Molecules of PIP2 form 6-nm diameter micelles in water, but aggregate into larger, multilamellar structures in physiological concentrations of divalent cations. Electron microscopic analysis of these aggregates reveals that they are clusters of striated filaments, suggesting that PIP2 aggregates form stacks of discoid micelles rather than multilamellar vesicles or inverted hexagonal arrays as previously inferred from indirect observations. The distance between striations within the filaments varies from 4.2 to 5.4 nm and the diameter of the filaments depends on the dehydrated ionic radius of the divalent cation, with average diameters of 19, 12, and 10 nm for filaments formed by Mg2+, Ca2+, and Ba2+, respectively. The structure of the divalent cation-induced aggregates can be altered by PIP2 binding proteins. Gelsolin and the microtubule associated protein tau both affect the formation of aggregates, indicating that tau acts as a PIP2 binding protein in a manner similar to gelsolin. In contrast, another PIP2 binding protein, profilin, does not modify the aggregates.