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1.
Commun Biol ; 5(1): 1211, 2022 11 10.
Artigo em Inglês | MEDLINE | ID: mdl-36357506

RESUMO

LRRK2 mutations are closely associated with Parkinson's disease (PD). Convergent evidence suggests that LRRK2 regulates striatal function. Here, by using knock-in mouse lines expressing the two most common LRRK2 pathogenic mutations-G2019S and R1441C-we investigated how LRRK2 mutations altered striatal physiology. While we found that both R1441C and G2019S mice displayed reduced nigrostriatal dopamine release, hypoexcitability in indirect-pathway striatal projection neurons, and alterations associated with an impaired striatal-dependent motor learning were observed only in the R1441C mice. We also showed that increased synaptic PKA activities in the R1441C and not G2019S mice underlie the specific alterations in motor learning deficits in the R1441C mice. In summary, our data argue that LRRK2 mutations' impact on the striatum cannot be simply generalized. Instead, alterations in electrochemical, electrophysiological, molecular, and behavioral levels were distinct between LRRK2 mutations. Our findings offer mechanistic insights for devising and optimizing treatment strategies for PD patients.


Assuntos
Corpo Estriado , Doença de Parkinson , Camundongos , Animais , Serina-Treonina Proteína Quinase-2 com Repetições Ricas em Leucina/genética , Serina-Treonina Proteína Quinase-2 com Repetições Ricas em Leucina/metabolismo , Corpo Estriado/metabolismo , Doença de Parkinson/patologia , Dopamina , Mutação
2.
J Neurosci ; 41(18): 4036-4059, 2021 05 05.
Artigo em Inglês | MEDLINE | ID: mdl-33731450

RESUMO

We have previously established that PV+ neurons and Npas1+ neurons are distinct neuron classes in the external globus pallidus (GPe): they have different topographical, electrophysiological, circuit, and functional properties. Aside from Foxp2+ neurons, which are a unique subclass within the Npas1+ class, we lack driver lines that effectively capture other GPe neuron subclasses. In this study, we examined the utility of Kcng4-Cre, Npr3-Cre, and Npy2r-Cre mouse lines (both males and females) for the delineation of GPe neuron subtypes. By using these novel driver lines, we have provided the most exhaustive investigation of electrophysiological studies of GPe neuron subtypes to date. Corroborating our prior studies, GPe neurons can be divided into two statistically distinct clusters that map onto PV+ and Npas1+ classes. By combining optogenetics and machine learning-based tracking, we showed that optogenetic perturbation of GPe neuron subtypes generated unique behavioral structures. Our findings further highlighted the dissociable roles of GPe neurons in regulating movement and anxiety-like behavior. We concluded that Npr3+ neurons and Kcng4+ neurons are distinct subclasses of Npas1+ neurons and PV+ neurons, respectively. Finally, by examining local collateral connectivity, we inferred the circuit mechanisms involved in the motor patterns observed with optogenetic perturbations. In summary, by identifying mouse lines that allow for manipulations of GPe neuron subtypes, we created new opportunities for interrogations of cellular and circuit substrates that can be important for motor function and dysfunction.SIGNIFICANCE STATEMENT Within the basal ganglia, the external globus pallidus (GPe) has long been recognized for its involvement in motor control. However, we lacked an understanding of precisely how movement is controlled at the GPe level as a result of its cellular complexity. In this study, by using transgenic and cell-specific approaches, we showed that genetically-defined GPe neuron subtypes have distinct roles in regulating motor patterns. In addition, the in vivo contributions of these neuron subtypes are in part shaped by the local, inhibitory connections within the GPe. In sum, we have established the foundation for future investigations of motor function and disease pathophysiology.


Assuntos
Globo Pálido/citologia , Globo Pálido/fisiologia , Atividade Motora/fisiologia , Neurônios/fisiologia , Animais , Ansiedade/psicologia , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Comportamento Animal , Fenômenos Biomecânicos , Fenômenos Eletrofisiológicos , Feminino , Aprendizado de Máquina , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Rede Nervosa/citologia , Rede Nervosa/fisiologia , Proteínas do Tecido Nervoso/genética , Optogenética , Canais de Potássio de Abertura Dependente da Tensão da Membrana/genética , Receptores do Fator Natriurético Atrial/genética
3.
J Neurosci ; 40(41): 7855-7876, 2020 10 07.
Artigo em Inglês | MEDLINE | ID: mdl-32868462

RESUMO

The external globus pallidus (GPe) is a critical node within the basal ganglia circuit. Phasic changes in the activity of GPe neurons during movement and their alterations in Parkinson's disease (PD) argue that the GPe is important in motor control. Parvalbumin-positive (PV+) neurons and Npas1+ neurons are the two principal neuron classes in the GPe. The distinct electrophysiological properties and axonal projection patterns argue that these two neuron classes serve different roles in regulating motor output. However, the causal relationship between GPe neuron classes and movement remains to be established. Here, by using optogenetic approaches in mice (both males and females), we showed that PV+ neurons and Npas1+ neurons promoted and suppressed locomotion, respectively. Moreover, PV+ neurons and Npas1+ neurons are under different synaptic influences from the subthalamic nucleus (STN). Additionally, we found a selective weakening of STN inputs to PV+ neurons in the chronic 6-hydroxydopamine lesion model of PD. This finding reinforces the idea that the reciprocally connected GPe-STN network plays a key role in disease symptomatology and thus provides the basis for future circuit-based therapies.SIGNIFICANCE STATEMENT The external pallidum is a key, yet an understudied component of the basal ganglia. Neural activity in the pallidum goes awry in neurologic diseases, such as Parkinson's disease. While this strongly argues that the pallidum plays a critical role in motor control, it has been difficult to establish the causal relationship between pallidal activity and motor function/dysfunction. This was in part because of the cellular complexity of the pallidum. Here, we showed that the two principal neuron types in the pallidum have opposing roles in motor control. In addition, we described the differences in their synaptic influence. Importantly, our research provides new insights into the cellular and circuit mechanisms that explain the hypokinetic features of Parkinson's disease.


Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Globo Pálido/fisiologia , Rede Nervosa/fisiologia , Proteínas do Tecido Nervoso/genética , Neurônios/fisiologia , Parvalbuminas/genética , Animais , Axônios/patologia , Fenômenos Eletrofisiológicos , Feminino , Globo Pálido/citologia , Locomoção/fisiologia , Masculino , Camundongos , Rede Nervosa/citologia , Optogenética , Núcleo Subtalâmico/citologia , Núcleo Subtalâmico/fisiologia , Sinapses/fisiologia
4.
J Neurosci ; 40(4): 743-768, 2020 01 22.
Artigo em Inglês | MEDLINE | ID: mdl-31811030

RESUMO

Within the basal ganglia circuit, the external globus pallidus (GPe) is critically involved in motor control. Aside from Foxp2+ neurons and ChAT+ neurons that have been established as unique neuron types, there is little consensus on the classification of GPe neurons. Properties of the remaining neuron types are poorly defined. In this study, we leverage new mouse lines, viral tools, and molecular markers to better define GPe neuron subtypes. We found that Sox6 represents a novel, defining marker for GPe neuron subtypes. Lhx6+ neurons that lack the expression of Sox6 were devoid of both parvalbumin and Npas1. This result confirms previous assertions of the existence of a unique Lhx6+ population. Neurons that arise from the Dbx1+ lineage were similarly abundant in the GPe and displayed a heterogeneous makeup. Importantly, tracing experiments revealed that Npas1+-Nkx2.1+ neurons represent the principal noncholinergic, cortically-projecting neurons. In other words, they form the pallido-cortical arm of the cortico-pallido-cortical loop. Our data further show that pyramidal-tract neurons in the cortex collateralized within the GPe, forming a closed-loop system between the two brain structures. Overall, our findings reconcile some of the discrepancies that arose from differences in techniques or the reliance on preexisting tools. Although spatial distribution and electrophysiological properties of GPe neurons reaffirm the diversification of GPe subtypes, statistical analyses strongly support the notion that these neuron subtypes can be categorized under the two principal neuron classes: PV+ neurons and Npas1+ neurons.SIGNIFICANCE STATEMENT The poor understanding of the neuronal composition in the external globus pallidus (GPe) undermines our ability to interrogate its precise behavioral and disease involvements. In this study, 12 different genetic crosses were used, hundreds of neurons were electrophysiologically characterized, and >100,000 neurons were histologically- and/or anatomically-profiled. Our current study further establishes the segregation of GPe neuron classes and illustrates the complexity of GPe neurons in adult mice. Our results support the idea that Npas1+-Nkx2.1+ neurons are a distinct GPe neuron subclass. By providing a detailed analysis of the organization of the cortico-pallidal-cortical projection, our findings establish the cellular and circuit substrates that can be important for motor function and dysfunction.


Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Córtex Cerebral/metabolismo , Globo Pálido/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Neurônios/metabolismo , Fator Nuclear 1 de Tireoide/metabolismo , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Camundongos , Camundongos Transgênicos , Proteínas do Tecido Nervoso/genética , Vias Neurais/metabolismo , Fator Nuclear 1 de Tireoide/genética
5.
J Neurosci ; 36(20): 5472-88, 2016 05 18.
Artigo em Inglês | MEDLINE | ID: mdl-27194328

RESUMO

UNLABELLED: Compelling evidence demonstrates that the external globus pallidus (GPe) plays a key role in processing sensorimotor information. An anatomical projection from the GPe to the dorsal striatum has been described for decades. However, the cellular target and functional impact of this projection remain unknown. Using cell-specific transgenic mice, modern monosynaptic tracing techniques, and optogenetics-based mapping, we discovered that GPe neurons provide inhibitory inputs to direct and indirect pathway striatal projection neurons (SPNs). Our results indicate that the GPe input to SPNs arises primarily from Npas1-expressing neurons and is strengthened in a chronic Parkinson's disease (PD) model. Alterations of the GPe-SPN input in a PD model argue for the critical position of this connection in regulating basal ganglia motor output and PD symptomatology. Finally, chemogenetic activation of Npas1-expressing GPe neurons suppresses motor output, arguing that strengthening of the GPe-SPN connection is maladaptive and may underlie the hypokinetic symptoms in PD. SIGNIFICANCE STATEMENT: An anatomical projection from the pallidum to the striatum has been described for decades, but little is known about its connectivity pattern. The authors dissect the presynaptic and postsynaptic neurons involved in this projection, and show its cell-specific remodeling and strengthening in parkinsonian mice. Chemogenetic activation of Npas1(+) pallidal neurons that give rise to the principal pallidostriatal projection increases the time that the mice spend motionless. This argues that maladaptive strengthening of this connection underlies the paucity of volitional movements, which is a hallmark of Parkinson's disease.


Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Globo Pálido/fisiologia , Proteínas do Tecido Nervoso/metabolismo , Neurônios/fisiologia , Potenciais Sinápticos , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Globo Pálido/citologia , Globo Pálido/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Atividade Motora , Proteínas do Tecido Nervoso/genética , Neurônios/metabolismo , Optogenética , Doença de Parkinson/metabolismo , Doença de Parkinson/patologia , Doença de Parkinson/fisiopatologia
6.
J Neurosci ; 35(16): 6584-99, 2015 Apr 22.
Artigo em Inglês | MEDLINE | ID: mdl-25904808

RESUMO

Striatal GABAergic interneurons that express the gene for tyrosine hydroxylase (TH) have been identified previously by several methods. Although generally assumed to be dopaminergic, possibly serving as a compensatory source of dopamine (DA) in Parkinson's disease, this assumption has never been tested directly. In TH-Cre mice whose nigrostriatal pathway had been eliminated unilaterally with 6-hydroxydopamine, we injected a Cre-dependent virus coding for channelrhodopsin-2 and enhanced yellow fluorescent protein unilaterally into the unlesioned midbrain or bilaterally into the striatum. Fast-scan cyclic voltammetry in striatal slices revealed that both optical and electrical stimulation readily elicited DA release in control striata but not from contralateral striata when nigrostriatal neurons were transduced. In contrast, neither optical nor electrical stimulation could elicit striatal DA release in either the control or lesioned striata when the virus was injected directly into the striatum transducing only striatal TH interneurons. This demonstrates that striatal TH interneurons do not release DA. Fluorescence immunocytochemistry in enhanced green fluorescent protein (EGFP)-TH mice revealed colocalization of DA, l-amino acid decarboxylase, the DA transporter, and vesicular monoamine transporter-2 with EGFP in midbrain dopaminergic neurons but not in any of the striatal EGFP-TH interneurons. Optogenetic activation of striatal EGFP-TH interneurons produced strong GABAergic inhibition in all spiny neurons tested. These results indicate that striatal TH interneurons are not dopaminergic but rather are a type of GABAergic interneuron that expresses TH but none of the other enzymes or transporters necessary to operate as dopaminergic neurons and exert widespread GABAergic inhibition onto direct and indirect spiny neurons.


Assuntos
Corpo Estriado/metabolismo , Neurônios Dopaminérgicos/metabolismo , Neurônios GABAérgicos/metabolismo , Interneurônios/metabolismo , Tirosina 3-Mono-Oxigenase/metabolismo , Animais , Descarboxilases de Aminoácido-L-Aromático/metabolismo , Corpo Estriado/fisiologia , Dopamina/metabolismo , Proteínas da Membrana Plasmática de Transporte de Dopamina/metabolismo , Estimulação Elétrica , Feminino , Neurônios GABAérgicos/fisiologia , Interneurônios/fisiologia , Masculino , Mesencéfalo/metabolismo , Camundongos , Camundongos Transgênicos , Inibição Neural/fisiologia , Optogenética , Estimulação Luminosa , Proteínas Vesiculares de Transporte de Monoamina/metabolismo
7.
Neuropharmacology ; 95: 468-76, 2015 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-25908399

RESUMO

The recent electrophysiological characterization of TH-expressing GABAergic interneurons (THINs) in the neostriatum revealed an unexpected degree of diversity of interneurons in this brain area (Ibáñez-Sandoval et al., 2010, Unal et al., 2011, 2015). Despite being relatively few in number, THINs may play a significant role in transmitting and distributing extra- and intrastriatal neuromodulatory signals in the striatal circuitry. Here we investigated the dopaminergic and cholinergic regulation of THINs in vitro. We found that the dominant effect of dopamine was a dramatic enhancement of the ability of THINs to generate long-lasting depolarizing plateau potentials (PPs). Interestingly, the same effect could also be elicited by amphetamine-induced release of endogenous dopamine suggesting that THINs may exhibit similar responses to changes in extracellular dopamine concentration in vivo. The enhancement of PPs in THINs is perhaps the most pronounced effect of dopamine on the intrinsic excitability of neostriatal neurons described to date. Further, we demonstrate that all subtypes of THINSs tested also express nicotinic cholinergic receptors. All THIS responded, albeit differentially, with depolarization, PPs and spiking to brief application of nicotinic agonists. Powerful modulation of the nonlinear integrative properties of THINs by dopamine and the direct depolarization of these neurons by acetylcholine may play important roles in mediating the effects of these neuromodulators in the neostriatum with potentially important implications for understanding the mechanisms of neuropsychiatric disorders affecting the basal ganglia.


Assuntos
Acetilcolina/metabolismo , Dopamina/metabolismo , Interneurônios/fisiologia , Neostriado/fisiologia , Tirosina 3-Mono-Oxigenase/metabolismo , Animais , Estimulação Elétrica , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Interneurônios/citologia , Interneurônios/efeitos dos fármacos , Potenciais da Membrana/efeitos dos fármacos , Potenciais da Membrana/fisiologia , Camundongos Transgênicos , Neostriado/citologia , Neostriado/efeitos dos fármacos , Agonistas Nicotínicos/farmacologia , Receptores de Dopamina D1/agonistas , Receptores de Dopamina D1/metabolismo , Receptores de Dopamina D5/agonistas , Receptores de Dopamina D5/metabolismo , Receptores Nicotínicos/metabolismo , Técnicas de Cultura de Tecidos , Tirosina 3-Mono-Oxigenase/genética
8.
J Neurosci ; 30(20): 7105-10, 2010 May 19.
Artigo em Inglês | MEDLINE | ID: mdl-20484653

RESUMO

Recent evidence suggests the intriguing possibility that midbrain dopaminergic (DAergic) neurons may use fast glutamatergic transmission to communicate with their postsynaptic targets. Because of technical limitations, direct demonstration of the existence of this signaling mechanism has been limited to experiments using cell culture preparations that often alter neuronal function including neurotransmitter phenotype. Consequently, it remains uncertain whether glutamatergic signaling between DAergic neurons and their postsynaptic targets exists under physiological conditions. Here, using an optogenetic approach, we provide the first conclusive demonstration that mesolimbic DAergic neurons in mice release glutamate and elicit excitatory postsynaptic responses in projection neurons of the nucleus accumbens. In addition, we describe the properties of the postsynaptic glutamatergic responses of these neurons during experimentally evoked burst firing of DAergic axons that reproduce the reward-related phasic population activity of the mesolimbic projection. These observations indicate that, in addition to DAergic mechanisms, mesolimbic reward signaling may involve glutamatergic transmission.


Assuntos
Dopamina/metabolismo , Ácido Glutâmico/metabolismo , Neurônios/metabolismo , Núcleo Accumbens/citologia , Transdução de Sinais/fisiologia , Animais , Dopaminérgicos/farmacologia , Estimulação Elétrica/métodos , Eletroquímica/métodos , Potenciais Pós-Sinápticos Excitadores/efeitos dos fármacos , Feminino , Técnicas de Transferência de Genes , Técnicas In Vitro , Proteínas Luminescentes/genética , Masculino , Camundongos , Neurônios/efeitos dos fármacos , Técnicas de Patch-Clamp/métodos , Estimulação Luminosa/métodos , Transdução de Sinais/efeitos dos fármacos , Bloqueadores dos Canais de Sódio/farmacologia , Transmissão Sináptica/efeitos dos fármacos , Transmissão Sináptica/fisiologia , Tetrodotoxina/farmacologia
9.
PLoS One ; 3(11): e3735, 2008.
Artigo em Inglês | MEDLINE | ID: mdl-19011687

RESUMO

Actin-based cell motility and force generation are central to immune response, tissue development, and cancer metastasis, and understanding actin cytoskeleton regulation is a major goal of cell biologists. Cell spreading is a commonly used model system for motility experiments -- spreading fibroblasts exhibit stereotypic, spatially-isotropic edge dynamics during a reproducible sequence of functional phases: 1) During early spreading, cells form initial contacts with the surface. 2) The middle spreading phase exhibits rapidly increasing attachment area. 3) Late spreading is characterized by periodic contractions and stable adhesions formation. While differences in cytoskeletal regulation between phases are known, a global analysis of the spatial and temporal coordination of motility and force generation is missing. Implementing improved algorithms for analyzing edge dynamics over the entire cell periphery, we observed that a single domain of homogeneous cytoskeletal dynamics dominated each of the three phases of spreading. These domains exhibited a unique combination of biophysical and biochemical parameters -- a motility module. Biophysical characterization of the motility modules revealed that the early phase was dominated by periodic, rapid membrane blebbing; the middle phase exhibited continuous protrusion with very low traction force generation; and the late phase was characterized by global periodic contractions and high force generation. Biochemically, each motility module exhibited a different distribution of the actin-related protein VASP, while inhibition of actin polymerization revealed different dependencies on barbed-end polymerization. In addition, our whole-cell analysis revealed that many cells exhibited heterogeneous combinations of motility modules in neighboring regions of the cell edge. Together, these observations support a model of motility in which regions of the cell edge exhibit one of a limited number of motility modules that, together, determine the overall motility function. Our data and algorithms are publicly available to encourage further exploration.


Assuntos
Membrana Celular/metabolismo , Movimento Celular , Fibroblastos/citologia , Animais , Apoptose/efeitos dos fármacos , Fenômenos Biomecânicos , Moléculas de Adesão Celular/metabolismo , Membrana Celular/efeitos dos fármacos , Movimento Celular/efeitos dos fármacos , Citocalasina D/farmacologia , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Camundongos , Proteínas dos Microfilamentos/metabolismo , Fosfoproteínas/metabolismo , Transporte Proteico/efeitos dos fármacos , Pseudópodes/efeitos dos fármacos , Pseudópodes/metabolismo
10.
Cell ; 129(4): 773-85, 2007 May 18.
Artigo em Inglês | MEDLINE | ID: mdl-17512410

RESUMO

The immunological synapse (IS) is a junction between the T cell and antigen-presenting cell and is composed of supramolecular activation clusters (SMACs). No studies have been published on naive T cell IS dynamics. Here, we find that IS formation during antigen recognition comprises cycles of stable IS formation and autonomous naive T cell migration. The migration phase is driven by PKCtheta, which is localized to the F-actin-dependent peripheral (p)SMAC. PKCtheta(-/-) T cells formed hyperstable IS in vitro and in vivo and, like WT cells, displayed fast oscillations in the distal SMAC, but they showed reduced slow oscillations in pSMAC integrity. IS reformation is driven by the Wiscott Aldrich Syndrome protein (WASp). WASp(-/-) T cells displayed normal IS formation but were unable to reform IS after migration unless PKCtheta was inhibited. Thus, opposing effects of PKCtheta and WASp control IS stability through pSMAC symmetry breaking and reformation.


Assuntos
Apresentação de Antígeno/fisiologia , Células Apresentadoras de Antígenos/metabolismo , Junções Intercelulares/metabolismo , Isoenzimas/metabolismo , Proteína Quinase C/metabolismo , Linfócitos T/metabolismo , Proteína da Síndrome de Wiskott-Aldrich/metabolismo , Animais , Células Apresentadoras de Antígenos/imunologia , Comunicação Celular/fisiologia , Movimento Celular/fisiologia , Ativação Enzimática/fisiologia , Inibidores Enzimáticos/farmacologia , Repressão Enzimática/efeitos dos fármacos , Repressão Enzimática/fisiologia , Junções Intercelulares/genética , Junções Intercelulares/imunologia , Isoenzimas/genética , Ativação Linfocitária/fisiologia , Lipídeos de Membrana/metabolismo , Camundongos , Camundongos Knockout , Proteína Quinase C/genética , Proteína Quinase C-theta , Linfócitos T/imunologia , Proteína da Síndrome de Wiskott-Aldrich/genética
11.
J Cell Sci ; 120(Pt 8): 1469-79, 2007 Apr 15.
Artigo em Inglês | MEDLINE | ID: mdl-17389687

RESUMO

Genetic or pharmacological alteration of the activity of the histone deacetylase 6 (HDAC6) induces a parallel alteration in cell migration. Using tubacin to block deacetylation of alpha-tubulin, and not other HDAC6 substrates, yielded a motility reduction equivalent to agents that block all NAD-independent HDACs. Accordingly, we investigated how the failure to deacetylate tubulin contributes to decreased motility in HDAC6-inhibited cells. Testing the hypothesis that motility is reduced because cellular adhesion is altered, we found that inhibiting HDAC6 activity towards tubulin rapidly increased total adhesion area. Next, we investigated the mechanism of the adhesion area increase. Formation of adhesions proceeded normally and cell spreading was more rapid in the absence of active HDAC6; however, photobleaching assays and adhesion breakdown showed that adhesion turnover was slower. To test the role of hyperacetylated tubulin in altering adhesion turnover, we measured microtubule dynamics in HDAC6-inhibited cells because dynamic microtubules are required to target adhesions for turnover. HDAC6 inhibition yielded a decrease in microtubule dynamics that was sufficient to decrease focal adhesion turnover. Thus, our results suggest a scenario in which the decreased dynamics of hyperacetylated microtubules in HDAC6-inhibited cells compromises their capacity to mediate the focal adhesion dynamics required for rapid cell migration.


Assuntos
Adesão Celular , Histona Desacetilases/metabolismo , Tubulina (Proteína)/metabolismo , Acetilação , Animais , Células COS , Movimento Celular , Chlorocebus aethiops , Desacetilase 6 de Histona , Humanos
12.
Phys Rev Lett ; 97(3): 038102, 2006 Jul 21.
Artigo em Inglês | MEDLINE | ID: mdl-16907546

RESUMO

We have monitored active movements of the cell circumference on specifically coated substrates for a variety of cells including mouse embryonic fibroblasts and T cells, as well as wing disk cells from fruit flies. Despite having different functions and being from multiple phyla, these cell types share a common spatiotemporal pattern in their normal membrane velocity; we show that protrusion and retraction events are organized in lateral waves along the cell membrane. These wave patterns indicate both spatial and temporal long-range periodic correlations of the actomyosin gel.


Assuntos
Membrana Celular/fisiologia , Movimento Celular/fisiologia , Fibroblastos/fisiologia , Linfócitos T/fisiologia , Actomiosina/química , Actomiosina/metabolismo , Animais , Drosophila melanogaster/citologia , Drosophila melanogaster/fisiologia , Fibroblastos/citologia , Géis/química , Camundongos , Modelos Biológicos , Linfócitos T/citologia , Fatores de Tempo
13.
J Cell Sci ; 119(Pt 7): 1307-19, 2006 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-16537651

RESUMO

R-Ras, an atypical member of the Ras subfamily of small GTPases, enhances integrin-mediated adhesion and signaling through a poorly understood mechanism. Dynamic analysis of cell spreading by total internal reflection fluorescence (TIRF) microscopy demonstrated that active R-Ras lengthened the duration of initial membrane protrusion, and promoted the formation of a ruffling lamellipod, rich in branched actin structures and devoid of filopodia. By contrast, dominant-negative R-Ras enhanced filopodia formation. Moreover, RNA interference (RNAi) approaches demonstrated that endogenous R-Ras contributed to cell spreading. These observations suggest that R-Ras regulates membrane protrusions through organization of the actin cytoskeleton. Our results suggest that phospholipase Cepsilon (PLCepsilon) is a novel R-Ras effector mediating the effects of R-Ras on the actin cytoskeleton and membrane protrusion, because R-Ras was co-precipitated with PLCepsilon and increased its activity. Knockdown of PLCepsilon with siRNA reduced the formation of the ruffling lamellipod in R-Ras cells. Consistent with this pathway, inhibitors of PLC activity, or chelating intracellular Ca2+ abolished the ability of R-Ras to promote membrane protrusions and spreading. Overall, these data suggest that R-Ras signaling regulates the organization of the actin cytoskeleton to sustain membrane protrusion through the activity of PLCepsilon.


Assuntos
Actinas/metabolismo , Pseudópodes/metabolismo , Fosfolipases Tipo C/metabolismo , Proteínas ras/metabolismo , Animais , Células COS , Cálcio/metabolismo , Adesão Celular , Linhagem Celular Transformada , Transformação Celular Viral , Quelantes/farmacologia , Chlorocebus aethiops , Relação Dose-Resposta a Droga , Ácido Egtázico/análogos & derivados , Ácido Egtázico/farmacologia , Feminino , Imunofluorescência , Corantes Fluorescentes , Proteínas de Fluorescência Verde/metabolismo , Humanos , Glândulas Mamárias Humanas/citologia , Microscopia de Fluorescência , Modelos Biológicos , Fosfoinositídeo Fosfolipase C , Testes de Precipitina , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Fosfolipases Tipo C/análise , Fosfolipases Tipo C/genética , Proteínas ras/genética
14.
Phys Rev Lett ; 93(10): 108105, 2004 Sep 03.
Artigo em Inglês | MEDLINE | ID: mdl-15447457

RESUMO

We monitored isotropic spreading of mouse embryonic fibroblasts on fibronectin-coated substrates. Cell adhesion area versus time was measured via total internal reflection fluorescence microscopy. Spreading proceeds in well-defined phases. We found a power-law area growth with distinct exponents in three sequential phases, which we denote as basal, continuous, and contractile spreading. High resolution differential interference contrast microscopy was used to characterize local membrane dynamics at the spreading front. Fourier power spectra of membrane velocity reveal the sudden development of periodic membrane retractions at the transition from continuous to contractile spreading. We propose that the classification of cell spreading into phases with distinct functional characteristics and protein activity serves as a paradigm for a general program of a phase classification of cellular phenotype.


Assuntos
Membrana Celular/fisiologia , Movimento Celular/fisiologia , Fibroblastos/citologia , Fibroblastos/fisiologia , Fluidez de Membrana/fisiologia , Modelos Biológicos , Proteínas Motores Moleculares/fisiologia , Animais , Adesão Celular/fisiologia , Divisão Celular/fisiologia , Células Cultivadas , Simulação por Computador , Fibronectinas/fisiologia , Camundongos
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