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1.
Proc Natl Acad Sci U S A ; 121(30): e2405114121, 2024 Jul 23.
Artigo em Inglês | MEDLINE | ID: mdl-39012825

RESUMO

Large cells often rely on cytoplasmic flows for intracellular transport, maintaining homeostasis, and positioning cellular components. Understanding the mechanisms of these flows is essential for gaining insights into cell function, developmental processes, and evolutionary adaptability. Here, we focus on a class of self-organized cytoplasmic stirring mechanisms that result from fluid-structure interactions between cytoskeletal elements at the cell cortex. Drawing inspiration from streaming flows in late-stage fruit fly oocytes, we propose an analytically tractable active carpet theory. This model deciphers the origins and three-dimensional spatiotemporal organization of such flows. Through a combination of simulations and weakly nonlinear theory, we establish the pathway of the streaming flow to its global attractor: a cell-spanning vortical twister. Our study reveals the inherent symmetries of this emergent flow, its low-dimensional structure, and illustrates how complex fluid-structure interaction aligns with classical solutions in Stokes flow. This framework can be easily adapted to elucidate a broad spectrum of self-organized, cortex-driven intracellular flows.


Assuntos
Citoplasma , Citoesqueleto , Animais , Citoplasma/metabolismo , Citoesqueleto/metabolismo , Modelos Biológicos , Oócitos/metabolismo , Corrente Citoplasmática/fisiologia
2.
J Cell Sci ; 136(5)2023 03 01.
Artigo em Inglês | MEDLINE | ID: mdl-36250267

RESUMO

Cells are the smallest building blocks of all living eukaryotic organisms, usually ranging from a couple of micrometers (for example, platelets) to hundreds of micrometers (for example, neurons and oocytes) in size. In eukaryotic cells that are more than 100 µm in diameter, very often a self-organized large-scale movement of cytoplasmic contents, known as cytoplasmic streaming, occurs to compensate for the physical constraints of large cells. In this Review, we discuss cytoplasmic streaming in multiple cell types and the mechanisms driving this event. We particularly focus on the molecular motors responsible for cytoplasmic movements and the biological roles of cytoplasmic streaming in cells. Finally, we describe bulk intercellular flow that transports cytoplasmic materials to the oocyte from its sister germline cells to drive rapid oocyte growth.


Assuntos
Proteínas de Drosophila , Cinesinas , Transporte Biológico/fisiologia , Corrente Citoplasmática/fisiologia , Proteínas de Drosophila/metabolismo , Dineínas/metabolismo , Microtúbulos/metabolismo , Oogênese
3.
Proc Natl Acad Sci U S A ; 119(8)2022 02 22.
Artigo em Inglês | MEDLINE | ID: mdl-35173046

RESUMO

Cytoplasmic streaming with extremely high velocity (∼70 µm s-1) occurs in cells of the characean algae (Chara). Because cytoplasmic streaming is caused by myosin XI, it has been suggested that a myosin XI with a velocity of 70 µm s-1, the fastest myosin measured so far, exists in Chara cells. However, the velocity of the previously cloned Chara corallina myosin XI (CcXI) was about 20 µm s-1, one-third of the cytoplasmic streaming velocity in Chara Recently, the genome sequence of Chara braunii has been published, revealing that this alga has four myosin XI genes. We cloned these four myosin XI (CbXI-1, 2, 3, and 4) and measured their velocities. While the velocities of CbXI-3 and CbXI-4 motor domains (MDs) were similar to that of CcXI MD, the velocities of CbXI-1 and CbXI-2 MDs were 3.2 times and 2.8 times faster than that of CcXI MD, respectively. The velocity of chimeric CbXI-1, a functional, full-length CbXI-1 construct, was 60 µm s-1 These results suggest that CbXI-1 and CbXI-2 would be the main contributors to cytoplasmic streaming in Chara cells and show that these myosins are ultrafast myosins with a velocity 10 times faster than fast skeletal muscle myosins in animals. We also report an atomic structure (2.8-Å resolution) of myosin XI using X-ray crystallography. Based on this crystal structure and the recently published cryo-electron microscopy structure of acto-myosin XI at low resolution (4.3-Å), it appears that the actin-binding region contributes to the fast movement of Chara myosin XI. Mutation experiments of actin-binding surface loops support this hypothesis.


Assuntos
Chara/genética , Corrente Citoplasmática/fisiologia , Miosinas/metabolismo , Actinas/metabolismo , Sequência de Aminoácidos , Microscopia Crioeletrônica , Corrente Citoplasmática/genética , Miosinas/genética
4.
Biophys J ; 122(2): 419-432, 2023 01 17.
Artigo em Inglês | MEDLINE | ID: mdl-36463405

RESUMO

Intercellular communication and transport is the essential prerequisite for the function of multicellular organisms. Simple diffusion as a transport mechanism is often inefficient in sustaining the effective exchange of metabolites, and other active transport mechanisms become involved. In this paper, we use the giant cells of characean algae as a model system to explore the role of advection and diffusion in intercellular transport. Using fluorescent dye as a tracer, we study the kinetics of the permeation of the fluorophore through the plasmodesmata complex in the node of tandem cells and its further distribution across the cell. To explore the role of cytoplasmic streaming and the nodal cell complex in the transport mechanism, we modulate the cytoplasmic streaming using action potential to separate the diffusive permeation from the advective contribution. The results imply that the plasmodesmal transport of fluorescent probe through the central and peripheral cells of the nodal complex is differentially regulated by a physiological signal, the action potential. The passage of the probe through the central cells of the nodal complex ceases transiently after elicitation of the action potential in the internodal cell, whereas the passage through the peripheral cells of the node was retained. A diffusion-advection model is developed to describe the transport kinetics and extract the permeability of the node-internode cell wall from experimental data.


Assuntos
Chara , Caráceas , Corantes Fluorescentes/metabolismo , Transporte Biológico , Corrente Citoplasmática/fisiologia
5.
Proc Natl Acad Sci U S A ; 114(8): E1385-E1394, 2017 02 21.
Artigo em Inglês | MEDLINE | ID: mdl-28096376

RESUMO

We investigate the myosin XI-driven transport network in Arabidopsis using protein-protein interaction, subcellular localization, gene knockout, and bioinformatics analyses. The two major groups of nodes in this network are myosins XI and their membrane-anchored receptors (MyoB) that, together, drive endomembrane trafficking and cytoplasmic streaming in the plant cells. The network shows high node connectivity and is dominated by generalists, with a smaller fraction of more specialized myosins and receptors. We show that interaction with myosins and association with motile vesicles are common properties of the MyoB family receptors. We identify previously uncharacterized myosin-binding proteins, putative myosin adaptors that belong to two unrelated families, with four members each (MadA and MadB). Surprisingly, MadA1 localizes to the nucleus and is rapidly transported to the cytoplasm, suggesting the existence of myosin XI-driven nucleocytoplasmic trafficking. In contrast, MadA2 and MadA3, as well as MadB1, partition between the cytosolic pools of motile endomembrane vesicles that colocalize with myosin XI-K and diffuse material that does not. Gene knockout analysis shows that MadB1-4 contribute to polarized root hair growth, phenocopying myosins, whereas MadA1-4 are redundant for this process. Phylogenetic analysis reveals congruent evolutionary histories of the myosin XI, MyoB, MadA, and MadB families. All these gene families emerged in green algae and show concurrent expansions via serial duplication in flowering plants. Thus, the myosin XI transport network increased in complexity and robustness concomitantly with the land colonization by flowering plants and, by inference, could have been a major contributor to this process.


Assuntos
Arabidopsis/metabolismo , Miosinas/metabolismo , Transporte Proteico/fisiologia , Proteínas de Arabidopsis/metabolismo , Corrente Citoplasmática/fisiologia , Filogenia , Raízes de Plantas/metabolismo , Receptores de Superfície Celular/metabolismo
6.
Biochem Biophys Res Commun ; 506(2): 403-408, 2018 11 25.
Artigo em Inglês | MEDLINE | ID: mdl-29307817

RESUMO

Actin is one of the three major cytoskeletal components in eukaryotic cells. Myosin XI is an actin-based motor protein in plant cells. Organelles are attached to myosin XI and translocated along the actin filaments. This dynamic actin-myosin XI system plays a major role in subcellular organelle transport and cytoplasmic streaming. Previous studies have revealed that myosin-driven transport and the actin cytoskeleton play essential roles in plant cell growth. Recent data have indicated that the actin-myosin XI cytoskeleton is essential for not only cell growth but also reproductive processes and responses to the environment. In this review, we have summarized previous reports regarding the role of the actin-myosin XI cytoskeleton in cytoplasmic streaming and plant development and recent advances in the understanding of the functions of actin-myosin XI cytoskeleton in Arabidopsis thaliana.


Assuntos
Citoesqueleto de Actina/metabolismo , Actinas/química , Proteínas de Arabidopsis/química , Arabidopsis/genética , Regulação da Expressão Gênica de Plantas , Miosinas/química , Citoesqueleto de Actina/genética , Citoesqueleto de Actina/ultraestrutura , Actinas/genética , Actinas/metabolismo , Arabidopsis/crescimento & desenvolvimento , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Fenômenos Biomecânicos , Corrente Citoplasmática/fisiologia , Regulação da Expressão Gênica no Desenvolvimento , Miosinas/genética , Miosinas/metabolismo , Especificidade de Órgãos , Células Vegetais/metabolismo , Células Vegetais/ultraestrutura , Folhas de Planta/genética , Folhas de Planta/crescimento & desenvolvimento , Folhas de Planta/metabolismo , Raízes de Plantas/genética , Raízes de Plantas/crescimento & desenvolvimento , Raízes de Plantas/metabolismo , Caules de Planta/genética , Caules de Planta/crescimento & desenvolvimento , Caules de Planta/metabolismo , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Reprodução
7.
Biophys J ; 113(1): 214-222, 2017 Jul 11.
Artigo em Inglês | MEDLINE | ID: mdl-28700920

RESUMO

The endoplasmic reticulum (ER) in plant cells forms a highly dynamic network of complex geometry. ER network morphology and dynamics are influenced by a number of biophysical processes, including filament/tubule tension, viscous forces, Brownian diffusion, and interactions with many other organelles and cytoskeletal elements. Previous studies have indicated that ER networks can be thought of as constrained minimal-length networks acted on by a variety of forces that perturb and/or remodel the network. Here, we study two specific biophysical processes involved in remodeling. One is the dynamic relaxation process involving a combination of tubule tension and viscous forces. The other is the rapid creation of cross-connection tubules by direct or indirect interactions with cytoskeletal elements. These processes are able to remodel the ER network: the first reduces network length and complexity whereas the second increases both. Using live cell imaging of ER network dynamics in tobacco leaf epidermal cells, we examine these processes on ER network dynamics. Away from regions of cytoplasmic streaming, we suggest that the dynamic network structure is a balance between the two processes, and we build an integrative model of the two processes for network remodeling. This model produces quantitatively similar ER networks to those observed in experiments. We use the model to explore the effect of parameter variation on statistical properties of the ER network.


Assuntos
Retículo Endoplasmático/metabolismo , Modelos Biológicos , Células Vegetais/metabolismo , Agrobacterium , Corrente Citoplasmática/fisiologia , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Microscopia Confocal , Folhas de Planta/citologia , Folhas de Planta/metabolismo , Análise de Célula Única , Nicotiana/citologia , Nicotiana/metabolismo , Transformação Genética , Proteína Vermelha Fluorescente
8.
BMC Cell Biol ; 18(1): 23, 2017 05 25.
Artigo em Inglês | MEDLINE | ID: mdl-28545392

RESUMO

BACKGROUND: In Dictyostelium discoideum, vesicular transport of the adenylyl cyclase A (ACA) to the posterior of polarized cells is essential to relay exogenous 3',5'-cyclic adenosine monophosphate (cAMP) signals during chemotaxis and for the collective migration of cells in head-to-tail arrangements called streams. RESULTS: Using fluorescence in situ hybridization (FISH), we discovered that the ACA mRNA is asymmetrically distributed at the posterior of polarized cells. Using both standard estimators and Monte Carlo simulation methods, we found that the ACA mRNA enrichment depends on the position of the cell within a stream, with the posterior localization of ACA mRNA being strongest for cells at the end of a stream. By monitoring the recovery of ACA-YFP after cycloheximide (CHX) treatment, we observed that ACA mRNA and newly synthesized ACA-YFP first emerge as fluorescent punctae that later accumulate to the posterior of cells. We also found that the ACA mRNA localization requires 3' ACA cis-acting elements. CONCLUSIONS: Together, our findings suggest that the asymmetric distribution of ACA mRNA allows the local translation and accumulation of ACA protein at the posterior of cells. These data represent a novel functional role for localized translation in the relay of chemotactic signal during chemotaxis.


Assuntos
Adenilil Ciclases , Quimiotaxia/genética , Dictyostelium/enzimologia , Proteínas de Protozoários , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Adenilil Ciclases/genética , Adenilil Ciclases/metabolismo , Animais , Polaridade Celular/efeitos dos fármacos , Polaridade Celular/genética , Células Cultivadas , Quimiotaxia/efeitos dos fármacos , Cicloeximida/farmacologia , Citoplasma/enzimologia , Corrente Citoplasmática/efeitos dos fármacos , Corrente Citoplasmática/fisiologia , Dictyostelium/metabolismo , Hibridização in Situ Fluorescente , Biossíntese de Proteínas/efeitos dos fármacos , Proteínas de Protozoários/genética , Proteínas de Protozoários/metabolismo , Transporte de RNA/fisiologia , RNA Mensageiro/análise , RNA de Protozoário/análise , RNA de Protozoário/genética , RNA de Protozoário/metabolismo , Sequências Reguladoras de Ácido Ribonucleico/fisiologia , Transdução de Sinais
9.
J Neurosci ; 33(17): 7439-50, 2013 Apr 24.
Artigo em Inglês | MEDLINE | ID: mdl-23616550

RESUMO

Alexander Disease (AxD) is a primary disorder of astrocytes, caused by heterozygous mutations in GFAP, which encodes the major astrocyte intermediate filament protein, glial fibrillary acidic protein (GFAP). Astrocytes in AxD display hypertrophy, massive increases in GFAP, and the accumulation of Rosenthal fibers, cytoplasmic protein inclusions containing GFAP, and small heat shock proteins. To study the effects of GFAP mutations on astrocyte morphology and physiology, we have examined hippocampal astrocytes in three mouse models of AxD, a transgenic line (GFAP(Tg)) in which the normal human GFAP is expressed in several copies, a knock-in line (Gfap(+/R236H)) in which one of the Gfap genes bears an R236H mutation, and a mouse derived from the mating of these two lines (GFAP(Tg); Gfap(+/R236H)). We report changes in astrocyte phenotype in all lines, with the most severe in the GFAP(Tg);Gfap(+/R236H), resulting in the conversion of protoplasmic astrocytes to cells that have lost their bushy-like morphology because of a reduction of distal fine processes, and become multinucleated and hypertrophic. Astrocytes activate the mTOR cascade, acquire CD44, and lose GLT-1. The altered astrocytes display a microheterogeneity in phenotypes, even neighboring cells. Astrocytes also show diminished glutamate transporter current, are significantly depolarized, and not coupled to adjacent astrocytes. Thus, the accumulation of GFAP in the AxD mouse astrocytes initiates a conversion of normal, protoplasmic astrocytes to astrocytes that display severely "reactive" characteristics, many of which may be detrimental to neighboring neurons and oligodendrocytes.


Assuntos
Doença de Alexander/genética , Doença de Alexander/patologia , Astrócitos/patologia , Corrente Citoplasmática/fisiologia , Modelos Animais de Doenças , Fenótipo , Doença de Alexander/metabolismo , Animais , Astrócitos/metabolismo , Hipocampo/metabolismo , Hipocampo/patologia , Camundongos , Camundongos Transgênicos , Técnicas de Cultura de Órgãos
10.
Development ; 138(6): 1087-92, 2011 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-21307100

RESUMO

The N-terminal head domain of kinesin heavy chain (Khc) is well known for generating force for transport along microtubules in cytoplasmic organization processes during metazoan development, but the functions of the C-terminal tail are not clear. To address this, we studied the effects of tail mutations on mitochondria transport, determinant mRNA localization and cytoplasmic streaming in Drosophila. Our results show that two biochemically defined elements of the tail - the ATP-independent microtubule-binding sequence and the IAK autoinhibitory motif - are essential for development and viability. Both elements have positive functions in the axonal transport of mitochondria and determinant mRNA localization in oocytes, processes that are accomplished by biased saltatory movement of individual cargoes. Surprisingly, there were no indications that the IAK autoinhibitory motif acts as a general downregulator of Kinesin-1 in those processes. Time-lapse imaging indicated that neither tail region is needed for fast cytoplasmic streaming in oocytes, which is a non-saltatory bulk transport process driven solely by Kinesin-1. Thus, the Khc tail is not constitutively required for Kinesin-1 activation, force transduction or linkage to cargo. It might instead be crucial for more subtle elements of motor control and coordination in the stop-and-go movements of biased saltatory transport.


Assuntos
Corrente Citoplasmática/genética , Proteínas de Drosophila/metabolismo , Cinesinas/metabolismo , Microtúbulos/metabolismo , Oócitos/metabolismo , Domínios e Motivos de Interação entre Proteínas/fisiologia , Sequência de Aminoácidos , Animais , Animais Geneticamente Modificados , Sítios de Ligação/fisiologia , Transporte Biológico/genética , Transporte Biológico/fisiologia , Corrente Citoplasmática/fisiologia , Drosophila/genética , Drosophila/metabolismo , Drosophila/fisiologia , Proteínas de Drosophila/química , Proteínas de Drosophila/genética , Proteínas de Drosophila/fisiologia , Retroalimentação Fisiológica/fisiologia , Feminino , Cinesinas/química , Cinesinas/genética , Cinesinas/fisiologia , Proteínas Associadas aos Microtúbulos/química , Proteínas Associadas aos Microtúbulos/metabolismo , Proteínas Associadas aos Microtúbulos/fisiologia , Dados de Sequência Molecular , Oócitos/fisiologia , Ligação Proteica/fisiologia , Domínios e Motivos de Interação entre Proteínas/genética
11.
Vis Neurosci ; 31(1): 1-10, 2014 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-24801619

RESUMO

Retinal pigment epithelial cells of teleosts contain numerous melanosomes (pigment granules) that exhibit light-dependent motility. In light, melanosomes disperse out of the retinal pigment epithelium (RPE) cell body (CB) into long apical projections that interdigitate with rod photoreceptors, thus shielding the photoreceptors from bleaching. In darkness, melanosomes aggregate through the apical projections back into the CB. Previous research has demonstrated that melanosome motility in the RPE CB requires microtubules, but in the RPE apical projections, actin filaments are necessary and sufficient for motility. We used myosin S1 labeling and platinum replica shadowing of dissociated RPE cells to determine actin filament polarity in apical projections. Actin filament bundles within RPE apical projections are uniformly oriented with barbed ends toward the distal tips. Treatment of RPE cells with the tetravalent lectin, Concanavalin A, which has been shown to suppress cortical actin flow by crosslinking of cell-surface proteins, inhibited melanosome aggregation and stimulated ectopic filopodia formation but did not block melanosome dispersion. The polarity orientation of F-actin in apical projections suggests that a barbed-end directed myosin motor could effect dispersion of melanosomes from the CB into apical projections. Inhibition of aggregation, but not dispersion, by ConA confirms that different actin-dependent mechanisms control these two processes and suggests that melanosome aggregation is sensitive to treatments previously shown to disrupt actin cortical flow.


Assuntos
Citoesqueleto de Actina/ultraestrutura , Concanavalina A/metabolismo , Melanossomas/fisiologia , Epitélio Pigmentado Ocular/citologia , Epitélio Pigmentado Ocular/metabolismo , Animais , Agregação Celular/fisiologia , Corrente Citoplasmática/fisiologia , Perciformes
12.
Proc Natl Acad Sci U S A ; 108(29): 11900-5, 2011 Jul 19.
Artigo em Inglês | MEDLINE | ID: mdl-21730185

RESUMO

Cytoplasmic streaming is a type of intracellular transport widely seen in nature. Cytoplasmic streaming in Caenorhabditis elegans at the one-cell stage is bidirectional; the flow near the cortex ("cortical flow") is oriented toward the anterior, whereas the flow in the central region ("cytoplasmic flow") is oriented toward the posterior. Both cortical flow and cytoplasmic flow depend on non-muscle-myosin II (NMY-2), which primarily localizes in the cortex. The manner in which NMY-2 proteins drive cytoplasmic flow in the opposite direction from remote locations has not been fully understood. In this study, we demonstrated that the hydrodynamic properties of the cytoplasm are sufficient to mediate the forces generated by the cortical myosin to drive bidirectional streaming throughout the cytoplasm. We quantified the flow velocities of cytoplasmic streaming using particle image velocimetry (PIV) and conducted a three-dimensional hydrodynamic simulation using the moving particle semiimplicit method. Our simulation quantitatively reconstructed the quantified flow velocity distribution resolved through PIV analysis. Furthermore, our PIV analyses detected microtubule-dependent flows during the pronuclear migration stage. These flows were reproduced via hydrodynamic interactions between moving pronuclei and the cytoplasm. The agreement of flow dynamics in vivo and in simulation indicates that the hydrodynamic properties of the cytoplasm are sufficient to mediate cytoplasmic streaming in C. elegans embryos.


Assuntos
Caenorhabditis elegans/fisiologia , Citoplasma/química , Corrente Citoplasmática/fisiologia , Embrião não Mamífero/fisiologia , Hidrodinâmica , Animais , Caenorhabditis elegans/embriologia , Proteínas de Caenorhabditis elegans/metabolismo , Simulação por Computador , Primers do DNA/genética , Microscopia Confocal , Microtúbulos/metabolismo , Cadeias Pesadas de Miosina/metabolismo , Interferência de RNA , Reologia
13.
Biofizika ; 59(6): 1143-50, 2014.
Artigo em Russo | MEDLINE | ID: mdl-25715623

RESUMO

In this study the experimental dependencies of the velocity of shuttle endoplasmic motion in the isolated plasmodial strand of Physarum polycephalum obtained by laser Doppler microscopy are presented. The spectral analysis of the time dependencies of the endoplasm allows obtaining two distinct harmonic components. Influence of KCN and SHAM--inhibitors of cellular respiration--leads to a complete cessation of endoplasmic motion in the strand. After removal of the inhibitors the respiratory system becomes normal, gradually restoring the activity of both harmonic oscillation sources. Based on the spectral analysis the simulated time-dependent velocity of the endoplasmic motion is rather good consistent with experimental data.


Assuntos
Relógios Biológicos/fisiologia , Corrente Citoplasmática/fisiologia , Citosol/metabolismo , Modelos Biológicos , Physarum polycephalum/metabolismo , Relógios Biológicos/efeitos dos fármacos , Corrente Citoplasmática/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Consumo de Oxigênio/efeitos dos fármacos , Consumo de Oxigênio/fisiologia , Physarum polycephalum/citologia , Cianeto de Potássio/farmacologia , Salicilamidas/farmacologia
14.
Plant Signal Behav ; 19(1): 2339574, 2024 Dec 31.
Artigo em Inglês | MEDLINE | ID: mdl-38601988

RESUMO

The giant (2-3 × 10-2 m long) internodal cells of the aquatic plant, Chara, exhibit a rapid (>100 × 10-6 m s-1) cyclic cytoplasmic streaming which stops in response to mechanical stimuli. Since the streaming - and the stopping of streaming upon stimulation - is easily visible with a stereomicroscope, these single cells are ideal tools to investigate mechanosensing in plant cells, as well as the potential for these cells to be anesthetized. We found that dropping a steel ball (0.88 × 10-3 kg, 6 × 10-3 m in diameter) through a 4.6 cm long tube (delivering ca. 4 × 10-4 J) reliably induced mechanically-stimulated cessation of cytoplasmic streaming. To determine whether mechanically-induced cessation of cytoplasmic streaming in Chara was sensitive to anesthesia, we treated Chara internodal cells to volatilized chloroform in a 9.8 × 10-3 m3 chamber for 2 minutes. We found that low doses (15,000-25,000 ppm) of chloroform did not always anesthetize cells, whereas large doses (46,000 and higher) proved lethal. However, 31,000 ppm chloroform completely, and reversibly, anesthetized these cells in that they did not stop cytoplasmic streaming upon mechanostimulation, but after 24 hours the cells recovered their sensitivity to mechanostimulation. We believe this single-cell model will prove useful for elucidating the still obscure mode of action of volatile anesthetics.


Assuntos
Anestesia , Chara , Clorofórmio , Corrente Citoplasmática/fisiologia
15.
Traffic ; 12(12): 1686-701, 2011 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-21920004

RESUMO

Organelle motility is an essential cellular function that is regulated by molecular motors, and their adaptors and activators. Here we established a new method that allows more direct investigation of the function of these peripheral membrane proteins in organelle motility than is possible by analysis of the organelle movement alone. This method uses multi-channel time-lapse microscopy to record the movement of organelles and associated fluorescent proteins, and automatic organelle tracking, to compare organelle movement parameters with the association of membrane proteins. This approach allowed large-scale, unbiased analysis of the contribution of organelle-associated proteins and cytoskeleton tracks in motility. Using this strategy, we addressed the role of membrane recruitment of Rab GTPases and effectors in organelle dynamics, using the melanosome as a model. We found that Rab27a and Rab32/38 were mainly recruited to sub-populations of slow-moving/static and fast-moving melanosomes, respectively. The correlation of Rab27a recruitment with slow movement/docking was dependent on the effector melanophilin. Meanwhile, using cytoskeleton-disrupting drugs, we observed that this speed:Rab content relationship corresponded to a decreased frequency of microtubule (MT)-based transport and an increased frequency of actin-dependent slow movement/docking. Overall, our data indicate the ability of Rab27a and effector recruitment to switch melanosomes from MT- to actin-based tethering and suggest that a network of Rab signalling may integrate melanosome biogenesis and transport.


Assuntos
Corrente Citoplasmática/fisiologia , Melanossomas/fisiologia , Proteínas de Membrana/metabolismo , Organelas/fisiologia , Proteínas rab de Ligação ao GTP/metabolismo , Actinas/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Animais , Células Cultivadas , Vesículas Citoplasmáticas/metabolismo , Vesículas Citoplasmáticas/fisiologia , Vetores Genéticos/genética , Melaninas/metabolismo , Melanócitos/metabolismo , Melanócitos/fisiologia , Melanossomas/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Microtúbulos/metabolismo , Organelas/metabolismo , Proteínas rab27 de Ligação ao GTP
16.
J Biol Chem ; 287(36): 30711-8, 2012 Aug 31.
Artigo em Inglês | MEDLINE | ID: mdl-22740687

RESUMO

Plant myosin XI functions as a motor that generates cytoplasmic streaming in plant cells. Although cytoplasmic streaming is known to be regulated by intracellular Ca(2+) concentration, the molecular mechanism underlying this control is not fully understood. Here, we investigated the mechanism of regulation of myosin XI by Ca(2+) at the molecular level. Actin filaments were easily detached from myosin XI in an in vitro motility assay at high Ca(2+) concentration (pCa 4) concomitant with the detachment of calmodulin light chains from the neck domains. Electron microscopic observations showed that myosin XI at pCa 4 shortened the neck domain by 30%. Single-molecule analysis revealed that the step size of myosin XI at pCa 4 was shortened to 27 nm under low load and to 22 nm under high load compared with 35 nm independent of the load for intact myosin XI. These results indicate that modulation of the mechanical properties of the neck domain is a key factor for achieving the Ca(2+)-induced regulation of cytoplasmic streaming.


Assuntos
Citoplasma/metabolismo , Corrente Citoplasmática/fisiologia , Miosinas/metabolismo , Nicotiana/enzimologia , Proteínas de Plantas/metabolismo , Citoesqueleto de Actina/genética , Citoesqueleto de Actina/metabolismo , Citoplasma/genética , Miosinas/genética , Proteínas de Plantas/genética , Estrutura Terciária de Proteína , Nicotiana/citologia , Nicotiana/genética
17.
Development ; 137(10): 1743-53, 2010 May.
Artigo em Inglês | MEDLINE | ID: mdl-20430749

RESUMO

In asymmetrically dividing cells, a failure to coordinate cell polarity with the site of cell division can lead to cell fate transformations and tumorigenesis. Cell polarity in C. elegans embryos is defined by PAR proteins, which occupy reciprocal halves of the cell cortex. During asymmetric division, the boundary between the anterior and posterior PAR domains precisely matches the site of cell division, ensuring exclusive segregation of cell fate. The PAR domains determine the site of cell division by positioning the mitotic spindle, suggesting one means by which cell polarity and cell division might be coordinated. Here, we report that cell polarity and cell division are coordinated through an additional mechanism: the site of cell division repositions the PAR-2 boundary. Galpha-mediated microtubule-cortex interactions appear to direct cortical flows of PAR-2 and myosin toward the site of cell division, which acts as a PAR-2 and myosin sink. Embryos with defects in PAR-2 boundary correction undergo mis-segregation of cortical polarity and cytoplasmic determinants, suggesting that PAR domain correction might help prevent cell fate transformation.


Assuntos
Padronização Corporal/fisiologia , Caenorhabditis elegans/embriologia , Divisão Celular/fisiologia , Polaridade Celular/fisiologia , Citocinese/fisiologia , Animais , Animais Geneticamente Modificados , Caenorhabditis elegans/fisiologia , Proteínas de Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/fisiologia , Fase de Clivagem do Zigoto/metabolismo , Fase de Clivagem do Zigoto/fisiologia , Corrente Citoplasmática/fisiologia , Embrião não Mamífero , Complexos Multiproteicos/metabolismo , Complexos Multiproteicos/fisiologia , Proteína Quinase C/metabolismo , Proteína Quinase C/fisiologia , Proteínas Serina-Treonina Quinases , Fuso Acromático/metabolismo
18.
Development ; 137(10): 1645-55, 2010 May.
Artigo em Inglês | MEDLINE | ID: mdl-20392741

RESUMO

Apical constriction is a major mechanism underlying tissue internalization during development. This cell constriction typically requires actomyosin contractility. Thus, understanding apical constriction requires characterization of the mechanics and regulation of actomyosin assemblies. We have analyzed the relationship between myosin and the polarity regulators Par-6, aPKC and Bazooka (Par-3) (the PAR complex) during amnioserosa apical constriction at Drosophila dorsal closure. The PAR complex and myosin accumulate at the apical surface domain of amnioserosa cells at dorsal closure, the PAR complex forming a patch of puncta and myosin forming an associated network. Genetic interactions indicate that the PAR complex supports myosin activity during dorsal closure, as well as during other steps of embryogenesis. We find that actomyosin contractility in amnioserosa cells is based on the repeated assembly and disassembly of apical actomyosin networks, with each assembly event driving constriction of the apical domain. As the networks assemble they translocate across the apical patch of PAR proteins, which persist at the apical domain. Through loss- and gain-of-function studies, we find that different PAR complex components regulate distinct phases of the actomyosin assembly/disassembly cycle: Bazooka promotes the duration of actomyosin pulses and Par-6/aPKC promotes the lull time between pulses. These results identify the mechanics of actomyosin contractility that drive amnioserosa apical constriction and how specific steps of the contractile mechanism are regulated by the PAR complex.


Assuntos
Actomiosina/fisiologia , Padronização Corporal/fisiologia , Drosophila/embriologia , Complexos Multiproteicos/fisiologia , Actinas/metabolismo , Actomiosina/genética , Actomiosina/metabolismo , Animais , Animais Geneticamente Modificados , Padronização Corporal/genética , Movimento Celular/genética , Movimento Celular/fisiologia , Polaridade Celular/genética , Polaridade Celular/fisiologia , Corrente Citoplasmática/fisiologia , Drosophila/genética , Drosophila/metabolismo , Drosophila/fisiologia , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Proteínas de Drosophila/fisiologia , Embrião não Mamífero , Peptídeos e Proteínas de Sinalização Intracelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/fisiologia , Proteínas de Membrana/metabolismo , Complexos Multiproteicos/genética , Complexos Multiproteicos/metabolismo , Ligação Proteica/fisiologia , Proteína Quinase C/genética , Proteína Quinase C/metabolismo , Proteína Quinase C/fisiologia , Multimerização Proteica/genética , Multimerização Proteica/fisiologia
19.
Proc Natl Acad Sci U S A ; 107(10): 4607-11, 2010 Mar 09.
Artigo em Inglês | MEDLINE | ID: mdl-20142479

RESUMO

A fundamental question in nutritional biology is how distributed systems maintain an optimal supply of multiple nutrients essential for life and reproduction. In the case of animals, the nutritional requirements of the cells within the body are coordinated by the brain in neural and chemical dialogue with sensory systems and peripheral organs. At the level of an insect society, the requirements for the entire colony are met by the foraging efforts of a minority of workers responding to cues emanating from the brood. Both examples involve components specialized to deal with nutrient supply and demand (brains and peripheral organs, foragers and brood). However, some of the most species-rich, largest, and ecologically significant heterotrophic organisms on earth, such as the vast mycelial networks of fungi, comprise distributed networks without specialized centers: How do these organisms coordinate the search for multiple nutrients? We address this question in the acellular slime mold Physarum polycephalum and show that this extraordinary organism can make complex nutritional decisions, despite lacking a coordination center and comprising only a single vast multinucleate cell. We show that a single slime mold is able to grow to contact patches of different nutrient quality in the precise proportions necessary to compose an optimal diet. That such organisms have the capacity to maintain the balance of carbon- and nitrogen-based nutrients by selective foraging has considerable implications not only for our understanding of nutrient balancing in distributed systems but for the functional ecology of soils, nutrient cycling, and carbon sequestration.


Assuntos
Carboidratos/farmacocinética , Physarum polycephalum/crescimento & desenvolvimento , Physarum polycephalum/metabolismo , Proteínas/farmacocinética , Animais , Carbono/metabolismo , Carbono/farmacocinética , Corrente Citoplasmática/fisiologia , Modelos Biológicos , Nitrogênio/metabolismo , Nitrogênio/farmacocinética , Fenômenos Fisiológicos da Nutrição , Physarum polycephalum/fisiologia
20.
J Cell Biol ; 176(7): 941-52, 2007 Mar 26.
Artigo em Inglês | MEDLINE | ID: mdl-17389229

RESUMO

Vesicular traffic in the Drosophila melanogaster oocyte occurs actively during vitellogenesis. Although endocytosis in the oocyte has been well characterized, exocytic vesicular traffic is less well understood. We show that the oocyte endoplasmic reticulum (ER) becomes concentrated into subcortical clusters during vitellogenesis. This ER reorganization requires Jagunal, which is an evolutionarily conserved ER membrane protein. Loss of Jagunal reduces vesicular traffic to the oocyte lateral membrane, but does not affect posterior polarized vesicular traffic, suggesting a role for Jagunal in facilitating vesicular traffic in the subcortex. Reduced membrane traffic caused by loss of Jagunal affects oocyte and bristle growth. We propose that ER reorganization is an important mechanism used by cells to prepare for an increased demand for membrane traffic, and Jagunal facilitates this process through ER clustering.


Assuntos
Proteínas de Drosophila/metabolismo , Drosophila melanogaster/metabolismo , Retículo Endoplasmático/metabolismo , Proteínas de Membrana/metabolismo , Oócitos/crescimento & desenvolvimento , Oogênese/fisiologia , Sequência de Aminoácidos , Animais , Sequência de Bases , Caenorhabditis elegans/genética , Diferenciação Celular/fisiologia , Sequência Conservada/genética , Corrente Citoplasmática/fisiologia , Proteínas de Drosophila/genética , Proteínas de Drosophila/isolamento & purificação , Drosophila melanogaster/ultraestrutura , Retículo Endoplasmático/ultraestrutura , Exocitose/fisiologia , Complexo de Golgi/metabolismo , Complexo de Golgi/ultraestrutura , Proteínas de Membrana/genética , Proteínas de Membrana/isolamento & purificação , Microscopia Eletrônica de Transmissão , Dados de Sequência Molecular , Oócitos/metabolismo , Oócitos/ultraestrutura , Transporte Proteico/fisiologia , Homologia de Sequência de Aminoácidos , Homologia de Sequência do Ácido Nucleico , Vesículas Transportadoras/metabolismo , Vesículas Transportadoras/ultraestrutura , Peixe-Zebra/genética
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