RESUMEN
Mapping 3D plasma membrane topology in live cells can bring unprecedented insights into cell biology. Widefield-based super-resolution methods such as 3D-structured illumination microscopy (3D-SIM) can achieve twice the axial ( ~ 300 nm) and lateral ( ~ 100 nm) resolution of widefield microscopy in real time in live cells. However, twice-resolution enhancement cannot sufficiently visualize nanoscale fine structures of the plasma membrane. Axial interferometry methods including fluorescence light interference contrast microscopy and its derivatives (e.g., scanning angle interference microscopy) can determine nanoscale axial locations of proteins on and near the plasma membrane. Thus, by combining super-resolution lateral imaging of 2D-SIM with axial interferometry, we developed multi-angle-crossing structured illumination microscopy (MAxSIM) to generate multiple incident angles by fast, optoelectronic creation of diffraction patterns. Axial localization accuracy can be enhanced by placing cells on a bottom glass substrate, locating a custom height-controlled mirror (HCM) at a fixed axial position above the glass substrate, and optimizing the height reconstruction algorithm for noisy experimental data. The HCM also enables imaging of both the apical and basal surfaces of a cell. MAxSIM with HCM offers high-fidelity nanoscale 3D topological mapping of cell plasma membranes with near-real-time ( ~ 0.5 Hz) imaging of live cells and 3D single-molecule tracking.
Asunto(s)
Algoritmos , Iluminación , Microscopía Fluorescente/métodos , Microscopía de Interferencia , InterferometríaRESUMEN
The Golgi complex comprises a connected ribbon of stacked cisternal membranes localized to the perinuclear region in most vertebrate cells. The position and morphology of this organelle depends upon interactions with microtubules and the actin cytoskeleton. In contrast, we know relatively little about the relationship of the Golgi complex with intermediate filaments (IFs). In this study, we show that the Golgi is in close physical proximity to vimentin IFs in cultured mouse and human cells. We also show that the trans-Golgi network coiled-coil protein GORAB can physically associate with vimentin IFs. Loss of vimentin and/or GORAB had a modest effect upon Golgi structure at the steady state. The Golgi underwent more rapid disassembly upon chemical disruption with brefeldin A or nocodazole, and slower reassembly upon drug washout, in vimentin knockout cells. Moreover, loss of vimentin caused reduced Golgi ribbon integrity when cells were cultured on high-stiffness hydrogels, which was exacerbated by loss of GORAB. These results indicate that vimentin IFs contribute to the structural stability of the Golgi complex and suggest a role for GORAB in this process.
Asunto(s)
Citoesqueleto , Filamentos Intermedios , Ratones , Humanos , Animales , Filamentos Intermedios/metabolismo , Vimentina/metabolismo , Citoesqueleto/metabolismo , Microtúbulos/metabolismo , Aparato de Golgi/metabolismo , Mamíferos/metabolismoRESUMEN
Protocols to assay the activity of glutamine synthetase (GS) are presented as they have been used in our laboratory to correlate the expression levels of the gene encoding Drosophila GS1 gene, the GS1 protein levels, and its activity in extracts of larvae and heads from Drosophila melanogaster. The assays are based on the glutamine synthetase-catalyzed formation of γ-glutamylhydroxylamine in the presence of ATP, L-glutamate, and hydroxylamine, in which hydroxylamine substitutes for ammonia in the reaction. Formation of γ-glutamylhydroxylamine is monitored spectrophotometrically in discontinuous assays upon complex formation with FeCl3. Fixed-time assays and those based on monitoring the time-course of product formation at different reaction times are described. The protocols can be adapted to quantify glutamine synthetase activity on biological materials other than Drosophila.
Asunto(s)
Drosophila melanogaster , Glutamato-Amoníaco Ligasa , Animales , Drosophila melanogaster/genética , Glutamato-Amoníaco Ligasa/genética , Glutamato-Amoníaco Ligasa/metabolismo , Ligasas , Drosophila/metabolismo , Ácido Glutámico , GlutaminaRESUMEN
Immunofluorescence is a technique that uses antibodies and fluorophores to label structures inside cells. The cells are normally fixed and permeabilized, and then structures are labelled using primary antibodies directly conjugated to fluorophores, or, more commonly, first with an antibody against an antigen of interest followed by a secondary antibody conjugated to a fluorophore that binds to the primary antibody. Fluorescence can be visualized using widefield, confocal, or super-resolution microscopy. Here we focus on labelling of the Golgi apparatus and show that different fixation and permeabilization conditions can significantly affect labelling of Golgi proteins and describe how to optimize fluorescent detection of Golgi proteins.
Asunto(s)
Colorantes Fluorescentes , Aparato de Golgi , Animales , Aparato de Golgi/metabolismo , Microscopía Fluorescente/métodos , Técnica del Anticuerpo Fluorescente , Colorantes Fluorescentes/metabolismo , Anticuerpos/metabolismo , Microscopía Confocal , MamíferosRESUMEN
Glutamine Synthetase 1 (GS1) is a key enzyme that catalyzes the ATP-dependent synthesis of l-glutamine from l-glutamate and is also member of the Glutamate Glutamine Cycle, a complex physiological process between glia and neurons that controls glutamate homeostasis and is often found compromised in neurodegenerative diseases including Huntington's disease (HD). Here we report that the expression of GS1 in neurons ameliorates the motility defects induced by the expression of the mutant Htt, using a Drosophila model for HD. This phenotype is associated with the ability of GS1 to favor the autophagy that we associate with the presence of reduced Htt toxic protein aggregates in neurons expressing mutant Htt. Expression of GS1 prevents the TOR activation and phosphorylation of S6K, a mechanism that we associate with the reduced levels of essential amino acids, particularly of arginine and asparagine important for TOR activation. This study reveals a novel function for GS1 to ameliorate neuronal survival by changing amino acids' levels that induce a "starvation-like" condition responsible to induce autophagy. The identification of novel targets that inhibit TOR in neurons is of particular interest for the beneficial role that autophagy has in preserving physiological neuronal health and in the mechanisms that eliminate the formation of toxic aggregates in proteinopathies.
Asunto(s)
Autofagia , Modelos Animales de Enfermedad , Glutamato-Amoníaco Ligasa/metabolismo , Enfermedad de Huntington/metabolismo , Enfermedad de Huntington/patología , Lisosomas/metabolismo , Neuronas/metabolismo , Animales , Drosophila melanogaster , Glutamato-Amoníaco Ligasa/genética , Enfermedad de Huntington/genética , Mutación , Neuronas/patologíaRESUMEN
ASAP1 is a multi-domain ArfGAP that controls cell migration, spreading, and focal adhesion dynamics. Although its GAP activity contributes to remodeling of the actin cytoskeleton, it does not fully explain all cellular functions of ASAP1. Here we find that ASAP1 regulates actin filament assembly directly through its N-BAR domain and controls stress fiber maintenance. ASAP1 depletion caused defects in stress fiber organization. Conversely, overexpression of ASAP1 enhanced actin remodeling. The BAR-PH fragment was sufficient to affect actin. ASAP1 with the BAR domain replaced with the BAR domain of the related ACAP1 did not affect actin. The BAR-PH tandem of ASAP1 bound and bundled actin filaments directly, whereas the presence of the ArfGAP and the C-terminal linker/SH3 domain reduced binding and bundling of filaments by BAR-PH. Together these data provide evidence that ASAP1 may regulate the actin cytoskeleton through direct interaction of the BAR-PH domain with actin filaments.
RESUMEN
ADP-ribosylation factors (Arfs) are members of the Ras GTPase superfamily. The function of Arfs is dependent on GTPase-activating proteins (GAPs) and guanine nucleotide exchange factors (GEFs), which allow Arfs to cycle between the GDP-bound and GTP-bound forms. Arf GAPs have been shown to be present in integrin adhesion complexes, which include focal adhesions. Integrin adhesion complexes are composed of integrins, scaffolding proteins and signaling proteins and regulate cell proliferation, survival, differentiation and migration. Understanding the role of Arf GAPs in the regulation of integrin adhesion complexes is relevant to understanding normal physiology and cancer. In this review, we will discuss the contribution of the Arf GAP family members to the regulation of integrin adhesion complexes, examining the diverse mechanisms by which they control integrin adhesion complex formation, maturation and dissolution. GIT1 and ARAP2 serve as GAPs for Arf6, regulating Rac1 and other effectors by mechanisms still being defined. In contrast, GIT2 regulates Rac1 independent of Arf6. AGAP2 binds to and regulates focal adhesion kinase (FAK). ARAP2 and ACAP1, both Arf6 GAPs, regulate membrane trafficking of integrins through different endocytic pathways, exerting opposite effects on focal adhesions. ASAP1 not only regulates actin cytoskeleton remodeling through its interaction with nonmuscle myosin 2A, but is also important in integrin recycling. These examples illustrate the diversity and versatility of Arf GAPs as regulators of integrin adhesion complex structure and function.
Asunto(s)
Factores de Ribosilacion-ADP/metabolismo , Adhesiones Focales/metabolismo , Neoplasias/metabolismo , Animales , Endocitosis , Humanos , Transporte de ProteínasRESUMEN
MICAL1 is a cytoplasmic 119 kDa protein participating in cytoskeleton dynamics through the NADPH-dependent oxidase and F-actin depolymerizing activities of its N-terminal flavoprotein domain, which is followed by calponin homology (CH), LIM domains and a C-terminal region with Pro-, Glu-rich and coiled-coil motifs. MICAL1 and truncated forms lacking the C-terminal, LIM and/or CH regions have been produced and characterized. The CH, LIM and C-terminal regions cause an increase of Km,NADPH exhibited by the NADPH oxidase activity of the flavoprotein domain, paralleling changes in the overall protein charge. The C-terminus also determines a â¼ 10-fold decrease of kcat, revealing its role in establishing an inactive/active conformational equilibrium, which is at the heart of the regulation of MICAL1 in cells. F-actin lowers Km,NADPH (10-50 µM) and increases kcat (10-25 s(-1)) to similar values for all MICAL forms. The apparent Km,actin of MICAL1 is â¼ 10-fold higher than that of the other forms (3-5 µM), reflecting the fact that F-actin binds to the flavoprotein domain in the MICAL's active conformation and stabilizes it. Analyses of the reaction in the presence of F-actin indicate that actin depolymerization is mediated by H2O2 produced by the NADPH oxidase reaction, rather than due to direct hydroxylation of actin methionine residues.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/química , Proteínas del Citoesqueleto/química , Citoesqueleto/química , Proteínas con Dominio LIM/química , Actinas/química , Animales , Biocatálisis , Humanos , Peróxido de Hidrógeno/química , Concentración de Iones de Hidrógeno , Cinética , Proteínas de Microfilamentos , Oxigenasas de Función Mixta , Modelos Moleculares , NADPH Oxidasas/química , Estructura Terciaria de Proteína , Conejos , Proteínas Recombinantes/química , ViscosidadRESUMEN
MICAL (from the Molecule Interacting with CasL) indicates a family of recently discovered cytosolic, multidomain proteins, which uniquely couple an N-terminal FAD-containing monooxygenase-like domain to typical calponine homology, LIM and coiled-coil protein-interaction modules. Genetic and cell biology approaches have demonstrated an essential role of the catalytic activity of the monooxygenase-like domain in transducing the signal initiated by semaphorins interaction with their plexin receptors, which results in local actin cytoskeleton disassembly as part of fundamental processes that include differentiation, migration and cell-cell contacts in neuronal and non-neuronal cell types. This review focuses on the structure-function relations of the MICAL monooxygenase-like domain as they are emerging from the available in vitro studies on mouse, human and Drosophila MICAL forms that demonstrated a NADPH-dependent actin depolymerizing activity of MICAL. With Drosophila MICAL forms, actin depolymerization was demonstrated to be associated to conversion of Met44 to methionine sulfone through a postulated hydroxylating reaction. Arguments supporting the concept that MICAL effect on F-actin may be reversible will be discussed.