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1.
Nature ; 609(7927): 575-581, 2022 09.
Artigo em Inglês | MEDLINE | ID: mdl-36071161

RESUMO

The phytohormone auxin triggers transcriptional reprogramming through a well-characterized perception machinery in the nucleus. By contrast, mechanisms that underlie fast effects of auxin, such as the regulation of ion fluxes, rapid phosphorylation of proteins or auxin feedback on its transport, remain unclear1-3. Whether auxin-binding protein 1 (ABP1) is an auxin receptor has been a source of debate for decades1,4. Here we show that a fraction of Arabidopsis thaliana ABP1 is secreted and binds auxin specifically at an acidic pH that is typical of the apoplast. ABP1 and its plasma-membrane-localized partner, transmembrane kinase 1 (TMK1), are required for the auxin-induced ultrafast global phospho-response and for downstream processes that include the activation of H+-ATPase and accelerated cytoplasmic streaming. abp1 and tmk mutants cannot establish auxin-transporting channels and show defective auxin-induced vasculature formation and regeneration. An ABP1(M2X) variant that lacks the capacity to bind auxin is unable to complement these defects in abp1 mutants. These data indicate that ABP1 is the auxin receptor for TMK1-based cell-surface signalling, which mediates the global phospho-response and auxin canalization.


Assuntos
Proteínas de Arabidopsis , Arabidopsis , Ácidos Indolacéticos , Proteínas Serina-Treonina Quinases , Arabidopsis/genética , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Corrente Citoplasmática , Concentração de Íons de Hidrogênio , Ácidos Indolacéticos/metabolismo , Mutação , Fosforilação , Reguladores de Crescimento de Plantas/metabolismo , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , ATPases Translocadoras de Prótons/metabolismo
2.
F S Sci ; 3(3): 210-216, 2022 08.
Artigo em Inglês | MEDLINE | ID: mdl-35661817

RESUMO

OBJECTIVE: To evaluate the developmental competency of mouse metaphase II oocytes and the pattern of mitochondrial positioning through cytoplasmic streaming in mouse metaphase II oocytes. DESIGN: We observed cytoplasmic streaming as movement indicated by fluorescently stained mitochondria using a newly developed method in which the spindle is translocated to the opposite site of the oocyte. This method is termed as intracytoplasmic spindle translocation (ICST). SETTING: University research laboratory. ANIMALS: Female B6D2F1 mice. INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): Fresh oocytes, postovulatory-aged oocytes, and oocytes treated with cytochalasin B were classified based on the presence of cytoplasmic streaming induced by ICST. The pattern of redistributed mitochondria and developmental competence caused by parthenogenetic activation were evaluated in oocytes with or without cytoplasmic streaming. RESULT(S): Induced cytoplasmic streaming occurred in 84% of the fresh oocytes but not in the postovulatory-aged oocytes and the oocytes treated with cytochalasin B. Abnormal mitochondrial aggregation was observed in oocytes in which cytoplasmic streaming was not induced. Furthermore, the developmental competence was significantly lower in oocytes without cytoplasmic streaming. CONCLUSION(S): Cytoplasmic streaming induced by ICST contributes to developmental competence through the redistribution of mitochondria and may be a valuable criterion for predicting early developmental competence in mouse oocytes.


Assuntos
Mitocôndrias , Oócitos , Animais , Citocalasina B/farmacologia , Corrente Citoplasmática , Feminino , Humanos , Camundongos , Partenogênese
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.
Sci Rep ; 12(1): 3150, 2022 02 24.
Artigo em Inglês | MEDLINE | ID: mdl-35210477

RESUMO

Arabidopsis thaliana has 13 genes belonging to the myosin XI family. Myosin XI-2 (MYA2) plays a major role in the generation of cytoplasmic streaming in Arabidopsis cells. In this study, we investigated the molecular properties of MYA2 expressed by the baculovirus transfer system. Actin-activated ATPase activity and in vitro motility assays revealed that activity of MYA2 was regulated by the globular tail domain (GTD). When the GTD is not bound to the cargo, the GTD inhibits ADP dissociation from the motor domain. Optical nanometry of single MYA2 molecules, combining total internal reflection fluorescence microscopy (TIRFM) and the fluorescence imaging with one-nanometer accuracy (FIONA) method, revealed that the MYA2 processively moved on actin with three different step sizes: - 28 nm, 29 nm, and 60 nm, at low ATP concentrations. This result indicates that MYA2 uses two different stepping modes; hand-over-hand and inchworm-like. Force measurement using optical trapping showed the stall force of MYA2 was 0.85 pN, which was less than half that of myosin V (2-3 pN). These results indicated that MYA2 has different transport properties from that of the myosin V responsible for vesicle transport in animal cells. Such properties may enable multiple myosin XIs to transport organelles quickly and smoothly, for the generation of cytoplasmic streaming in plant cells.


Assuntos
Arabidopsis/metabolismo , Corrente Citoplasmática , Cadeias Pesadas de Miosina/metabolismo , Organelas/metabolismo , Arabidopsis/genética , Cadeias Pesadas de Miosina/genética , Organelas/genética
5.
Mol Biol Cell ; 32(5): 413-421, 2021 03 01.
Artigo em Inglês | MEDLINE | ID: mdl-33405963

RESUMO

Uveal melanomas (UMs) are malignant cancers arising from the pigmented layers of the eye. UM cells spread through the bloodstream, and circulating UM cells are detectable in patients before metastases appear. Extravasation of UM cells is necessary for formation of metastases, and transendothelial migration (TEM) is a key step in extravasation. UM cells execute TEM via a stepwise process involving the actin-based processes of ameboid blebbing and mesenchymal lamellipodial protrusion. UM cancers are driven by oncogenic mutations that activate Gαq/11, and this activates TRIO, a guanine nucleotide exchange factor for RhoA and Rac1. We found that pharmacologic inhibition of Gαq/11 in UM cells reduced TEM. Inhibition of the RhoA pathway blocked amoeboid motility but led to enhanced TEM; in contrast, inhibition of the Rac1 pathway decreased mesenchymal motility and reduced TEM. Inhibition of Arp2/3 complex allowed cells to transmigrate without intercalation, a direct mechanism similar to the one often displayed by immune cells. BAP1-deficient (+/-) UM subclones displayed motility behavior and increased levels of TEM, similar to the effects of RhoA inhibitors. We conclude that RhoA and Rac1 signaling pathways, downstream of oncogenic Gαq/11, combine with pathways regulated by BAP1 to control the motility and transmigration of UM cells.


Assuntos
Movimento Celular/fisiologia , Melanoma/metabolismo , Migração Transendotelial e Transepitelial/fisiologia , Neoplasias Uveais/metabolismo , Vesícula/metabolismo , Linhagem Celular Tumoral , Corrente Citoplasmática/fisiologia , Endotélio/metabolismo , Endotélio/patologia , Subunidades alfa Gq-G11 de Proteínas de Ligação ao GTP/metabolismo , Humanos , Melanoma/patologia , Pseudópodes/metabolismo , Transdução de Sinais/genética , Proteínas Supressoras de Tumor/metabolismo , Ubiquitina Tiolesterase/metabolismo , Neoplasias Uveais/patologia , Proteínas rac1 de Ligação ao GTP/metabolismo , Proteína rhoA de Ligação ao GTP/metabolismo
6.
Phys Rev Lett ; 126(2): 028103, 2021 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-33512217

RESUMO

In the cellular phenomena of cytoplasmic streaming, molecular motors carrying cargo along a network of microtubules entrain the surrounding fluid. The piconewton forces produced by individual motors are sufficient to deform long microtubules, as are the collective fluid flows generated by many moving motors. Studies of streaming during oocyte development in the fruit fly Drosophila melanogaster have shown a transition from a spatially disordered cytoskeleton, supporting flows with only short-ranged correlations, to an ordered state with a cell-spanning vortical flow. To test the hypothesis that this transition is driven by fluid-structure interactions, we study a discrete-filament model and a coarse-grained continuum theory for motors moving on a deformable cytoskeleton, both of which are shown to exhibit a swirling instability to spontaneous large-scale rotational motion, as observed.


Assuntos
Citoesqueleto/química , Citoesqueleto/metabolismo , Microtúbulos/química , Microtúbulos/metabolismo , Modelos Biológicos , Animais , Fenômenos Biomecânicos , Citoplasma/química , Citoplasma/metabolismo , Corrente Citoplasmática , Drosophila melanogaster
7.
Mol Biol Cell ; 31(16): 1765-1773, 2020 07 21.
Artigo em Inglês | MEDLINE | ID: mdl-32459552

RESUMO

Cell polarization is required to define body axes during development. The position of spatial cues for polarization is critical to direct the body axes. In Caenorhabditis elegans zygotes, the sperm-derived pronucleus/centrosome complex (SPCC) serves as the spatial cue to specify the anterior-posterior axis. Approximately 30 min after fertilization, the contractility of the cell cortex is relaxed near the SPCC, which is the earliest sign of polarization and called symmetry breaking (SB). It is unclear how the position of SPCC at SB is determined after fertilization. Here, we show that SPCC drifts dynamically through the cell-wide flow of the cytoplasm, called meiotic cytoplasmic streaming. This flow occasionally brings SPCC to the opposite side of the sperm entry site before SB. Our results demonstrate that cytoplasmic flow determines stochastically the position of the spatial cue of the body axis, even in an organism like C. elegans for which development is stereotyped.


Assuntos
Polaridade Celular/fisiologia , Corrente Citoplasmática/fisiologia , Zigoto/metabolismo , Animais , Padronização Corporal/fisiologia , Caenorhabditis elegans/embriologia , Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/metabolismo , Centrossomo/metabolismo , Citoplasma/fisiologia , Fertilização/fisiologia , Masculino , Espermatozoides/metabolismo
8.
Mol Biol Cell ; 31(12): 1246-1258, 2020 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-32267197

RESUMO

The orientation of microtubule (MT) networks is exploited by motors to deliver cargoes to specific intracellular destinations and is thus essential for cell polarity and function. Reconstituted in vitro systems have largely contributed to understanding the molecular framework regulating the behavior of MT filaments. In cells, however, MTs are exposed to various biomechanical forces that might impact on their orientation, but little is known about it. Oocytes, which display forceful cytoplasmic streaming, are excellent model systems to study the impact of motion forces on cytoskeletons in vivo. Here we implement variational optical flow analysis as a new approach to analyze the polarity of MTs in the Drosophila oocyte, a cell that displays distinct Kinesin-dependent streaming. After validating the method as robust for describing MT orientation from confocal movies, we find that increasing the speed of flows results in aberrant plus end growth direction. Furthermore, we find that in oocytes where Kinesin is unable to induce cytoplasmic streaming, the growth direction of MT plus ends is also altered. These findings lead us to propose that cytoplasmic streaming - and thus motion by advection - contributes to the correct orientation of MTs in vivo. Finally, we propose a possible mechanism for a specialized cytoplasmic actin network (the actin mesh) to act as a regulator of flow speeds to counteract the recruitment of Kinesin to MTs.


Assuntos
Cinesinas/metabolismo , Microtúbulos/fisiologia , Oócitos/metabolismo , Actinas/metabolismo , Animais , Fenômenos Biomecânicos , Polaridade Celular , Citoplasma/metabolismo , Corrente Citoplasmática/fisiologia , Citoesqueleto/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/metabolismo , Dineínas/metabolismo , Feminino , Cinesinas/fisiologia , Fenômenos Mecânicos , Microtúbulos/metabolismo , Fluxo Óptico , Orientação Espacial/fisiologia
9.
Dev Cell ; 51(2): 135-144, 2019 10 21.
Artigo em Inglês | MEDLINE | ID: mdl-31639366

RESUMO

Spatiotemporal organization during development has frequently been proposed to be explainable by reaction-transport models, where biochemical reactions couple to physical motion. However, whereas genetic tools allow causality of molecular players to be dissected via perturbation experiments, the functional role of physical transport processes, such as diffusion and cytoplasmic streaming, frequently remains untestable. This Perspective explores the challenges of validating reaction-transport hypotheses and highlights new opportunities provided by perturbation approaches that specifically target physical transport mechanisms. Using these methods, experimental physics may begin to catch up with molecular biology and find ways to test roles of diffusion and flows in development.


Assuntos
Transporte Biológico/fisiologia , Fenômenos Fisiológicos Celulares/fisiologia , Corrente Citoplasmática/fisiologia , Movimento (Física) , Animais , Difusão , Humanos , Modelos Biológicos
10.
Cell Biochem Biophys ; 77(4): 357-366, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31562588

RESUMO

This study aimed to investigate for the first time, the profile of Physarum microplasmodial phosphatase (PPH) activity toward the phosphorylated light chain of Physarum myosin II (PLCM) at pH 7.6, the velocity of cytoplasmic streaming, and PPH expression in spherule formation during dark starvation (DS). In this study, we cloned the full-length cDNA of PPH using polymerase chain reaction, based on the N-terminal amino acid sequence of the purified enzyme. The cDNA contained an open reading frame (ORF) of 1245 bp, corresponding to 415 amino acids. We confirmed that a rapid increase in PPH activity toward PLCM and a rapid decrease in cytoplasmic streaming velocity precede spherule formation by Physarum microplasmodia. The profiles of increase in PPH activity toward PLCM, PPH expression, and PPH accumulation during DS were correlated with spherule formation in the Physarum microplasmodia. Moreover, application of the wheat germ cell-free expression system resulted in the successful production of recombinant PPH and in the expression of phosphatase activity toward PLCM. These results suggest that PPH is involved in the cessation of cytoplasmic streaming in Physarum microplasmodia during DS.


Assuntos
Corrente Citoplasmática/fisiologia , Monoéster Fosfórico Hidrolases/metabolismo , Physarum/enzimologia , Proteínas de Protozoários/metabolismo , Sequência de Aminoácidos , Clonagem Molecular , Miosina Tipo II/metabolismo , Monoéster Fosfórico Hidrolases/química , Monoéster Fosfórico Hidrolases/genética , Fosforilação , Proteínas de Protozoários/química , Proteínas de Protozoários/genética , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação
11.
Bioelectrochemistry ; 129: 62-69, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31103848

RESUMO

Immobile chloroplasts in Chara internodal cells release photometabolites into the streaming cytoplasm that distributes the exported solutes and provides metabolic connectivity between spatially remote plastids. The metabolite transmission by fluid flow is evident from chlorophyll fluorescence changes in shaded chloroplasts upon local illumination applied upstream of the analyzed area. The connectivity correlates with the pH pattern on cell surface: it is strong in cell regions with high H+-pump activity and is low in regions featuring large passive H+ influx (OH- efflux). One explanation for low connectivity under the alkaline bands is that H+ influx lowers the cytoplasmic pH, thus retarding metabolic conversions of solutes carried by the microfluidic transporter. The cessation of H+ influx across the plasma membrane by eliciting the action potential and by adding NH4Cl into the medium greatly enhanced the amplitude of cyclosis-mediated fluorescence transients. The transition from latent to the transmissive state after the dark pretreatment was paralleled by the temporary increase in chlorophyll fluorescence, reflecting changes in photosynthetic electron transport. It is proposed that the connectivity between distant chloroplasts is controlled by cytoplasmic pH.


Assuntos
Membrana Celular/metabolismo , Chara/citologia , Cloroplastos/metabolismo , Corrente Citoplasmática , Comunicação Celular , Chara/metabolismo , Concentração de Íons de Hidrogênio , Luz , Prótons
12.
Protoplasma ; 256(3): 815-826, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-30610387

RESUMO

Symplastic interconnections of plant cells via perforations in adjoining cell walls (plasmodesmata) enable long-distance transport of photoassimilates and signaling substances required for growth and development. The pathways and features of intercellular movement of assimilates are often examined with fluorescent tracers whose molecular dimensions are similar to natural metabolites produced in photosynthesis. Chlorophyll fluorescence was recently found to be a sensitive noninvasive indicator of long-distance intracellular transport of physiologically produced photometabolites in characean internodes. The present work shows that the chlorophyll microfluorometry has a potential for studying the cell-to-cell transport of reducing substances released by local illumination of one internode and detected as the fluorescence increase in the neighbor internode. The method provides temporal resolution in the time frame of seconds and can be used to evaluate permeability of plasmodesmata to natural components released by illuminated chloroplasts. The results show that approximately one third of the amount of photometabolites released into the streaming cytoplasm during a 30-s pulse of local light permeates across the nodal complex with the characteristic time of ~ 10 s. The intercellular transport was highly sensitive to moderate elevations of osmolarity in the bath solution (150 mM sorbitol), which contrasts to the view that only transnodal gradients in osmolarity (and internal hydrostatic pressure) have an appreciable influence on plasmodesmal conductance. The inhibition of cell-to-cell transport was reversible and specific; the sorbitol addition had no influence on photosynthetic electron transport and the velocity of cytoplasmic streaming. The conductance of transcellular pores increased in the presence of the actin inhibitor cytochalasin D but the cell-to-cell transport was eventually suppressed due to the deceleration and cessation of cytoplasmic streaming. The results show that the permeability of plasmodesmata to low-molecular photometabolites is subject to upregulation and downregulation.


Assuntos
Chara/fisiologia , Clorofila/metabolismo , Citofotometria/métodos , Corrente Citoplasmática , Fotossíntese , Cátions Bivalentes/farmacologia , Chara/efeitos dos fármacos , Citocalasina D/farmacologia , Corrente Citoplasmática/efeitos dos fármacos , Desidratação , Fluorescência , Concentração de Íons de Hidrogênio , Ionóforos/farmacologia , Metaboloma/efeitos dos fármacos , Osmose/efeitos dos fármacos , Fotossíntese/efeitos dos fármacos , Prótons
13.
Elife ; 82019 01 02.
Artigo em Inglês | MEDLINE | ID: mdl-30601119

RESUMO

We introduce a filament-based simulation model for coarse-grained, effective motor-mediated interaction between microtubule pairs to study the time-scales that compose cytoplasmic streaming. We characterise microtubule dynamics in two-dimensional systems by chronologically arranging five distinct processes of varying duration that make up streaming, from microtubule pairs to collective dynamics. The structures found were polarity sorted due to the propulsion of antialigned microtubules. This also gave rise to the formation of large polar-aligned domains, and streaming at the domain boundaries. Correlation functions, mean squared displacements, and velocity distributions reveal a cascade of processes ultimately leading to microtubule streaming and advection, spanning multiple microtubule lengths. The characteristic times for the processes extend over three orders of magnitude from fast single-microtubule processes to slow collective processes. Our approach can be used to directly test the importance of molecular components, such as motors and crosslinking proteins between microtubules, on the collective dynamics at cellular scale.


Assuntos
Citoplasma/metabolismo , Corrente Citoplasmática , Microtúbulos/metabolismo , Animais , Simulação por Computador , Citoesqueleto/metabolismo , Drosophila , Feminino , Cinesinas/metabolismo , Microscopia de Vídeo , Oócitos/metabolismo , Fatores de Tempo
14.
Soft Matter ; 15(2): 190-199, 2019 Jan 02.
Artigo em Inglês | MEDLINE | ID: mdl-30488938

RESUMO

During physiological processes, cells can undergo morphological changes that can result in a significant redistribution of the cytoskeleton causing anisotropic behavior. Evidence of anisotropy in cells under mechanical stimuli exists; however, the role of cytoskeletal restructuring resulting from changes in cell shape in mechanical anisotropy and its effects remain unclear. In the present study, we examine the role of cell morphology in inducing anisotropy in both intracellular mechanics and dynamics. We change the aspect ratio of cells by confining the cell width and measuring the mechanical properties of the cytoplasm using optical tweezers in both the longitudinal and transverse directions to quantify the degree of mechanical anisotropy. These active microrheology measurements are then combined with intracellular movement to calculate the intracellular force spectrum using force spectrum microscopy (FSM), from which the degree of anisotropy in dynamics and force can be quantified. We find that unrestricted cells with aspect ratio (AR) ∼1 are isotropic; however, when cells break symmetry, they exhibit significant anisotropy in cytoplasmic mechanics and dynamics.


Assuntos
Citoplasma/ultraestrutura , Corrente Citoplasmática , Citoesqueleto/ultraestrutura , Fibroblastos/citologia , Animais , Anisotropia , Fenômenos Biomecânicos , Linhagem Celular , Forma Celular , Tamanho Celular , Citoplasma/metabolismo , Citoesqueleto/metabolismo , Fibroblastos/metabolismo , Camundongos , Movimento (Física) , Pinças Ópticas , Reologia
15.
Nat Commun ; 9(1): 4694, 2018 11 08.
Artigo em Inglês | MEDLINE | ID: mdl-30410005

RESUMO

Syncytial architecture is an evolutionarily-conserved feature of the germline of many species and plays a crucial role in their fertility. However, the mechanism supporting syncytial organization is largely unknown. Here, we identify a corset-like actomyosin structure within the syncytial germline of Caenorhabditis elegans, surrounding the common rachis. Using laser microsurgery, we demonstrate that actomyosin contractility within this structure generates tension both in the plane of the rachis surface and perpendicular to it, opposing membrane tension. Genetic and pharmacological perturbations, as well as mathematical modeling, reveal a balance of forces within the gonad and show how changing the tension within the actomyosin corset impinges on syncytial germline structure, leading, in extreme cases, to sterility. Thus, our work highlights a unique tissue-level cytoskeletal structure, and explains the critical role of actomyosin contractility in the preservation of a functional germline.


Assuntos
Actomiosina/metabolismo , Células Germinativas/metabolismo , Células Gigantes/metabolismo , Animais , Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/metabolismo , Corrente Citoplasmática , Gônadas/metabolismo , Modelos Biológicos , Miosinas/metabolismo
16.
Elife ; 72018 07 25.
Artigo em Inglês | MEDLINE | ID: mdl-30044219

RESUMO

Regulating nuclear histone balance is essential for survival, yet in early Drosophila melanogaster embryos many regulatory strategies employed in somatic cells are unavailable. Previous work had suggested that lipid droplets (LDs) buffer nuclear accumulation of the histone variant H2Av. Here, we elucidate the buffering mechanism and demonstrate that it is developmentally controlled. Using live imaging, we find that H2Av continuously exchanges between LDs. Our data suggest that the major driving force for H2Av accumulation in nuclei is H2Av abundance in the cytoplasm and that LD binding slows nuclear import kinetically, by limiting this cytoplasmic pool. Nuclear H2Av accumulation is indeed inversely regulated by overall buffering capacity. Histone exchange between LDs abruptly ceases during the midblastula transition, presumably to allow canonical regulatory mechanisms to take over. These findings provide a mechanistic basis for the emerging role of LDs as regulators of protein homeostasis and demonstrate that LDs can control developmental progression.


Assuntos
Proteínas de Drosophila/genética , Drosophila melanogaster/embriologia , Drosophila melanogaster/genética , Embrião não Mamífero/metabolismo , Histonas/genética , Gotículas Lipídicas/metabolismo , Transporte Ativo do Núcleo Celular , Animais , Blastoderma/metabolismo , Núcleo Celular/metabolismo , Cromossomos/metabolismo , Corrente Citoplasmática , Proteínas de Drosophila/metabolismo , Desenvolvimento Embrionário , Dosagem de Genes , Regulação da Expressão Gênica no Desenvolvimento , Histonas/metabolismo , Interfase , Cinética , Modelos Biológicos
17.
Artigo em Inglês | MEDLINE | ID: mdl-29496823

RESUMO

The delivery of intracellular material within cells is crucial for maintaining normal function. Myosins transport a wide variety of cargo, ranging from vesicles to ribonuclear protein particles (RNPs), in plants, fungi, and metazoa. The properties of a given myosin transporter are adapted to move on different actin filament tracks, either on the disordered actin networks at the cell cortex or along highly organized actin bundles to distribute their cargo in a localized manner or move it across long distances in the cell. Transport is controlled by selective recruitment of the myosin to its cargo that also plays a role in activation of the motor.


Assuntos
Miosinas/metabolismo , Animais , Transporte Biológico , Corrente Citoplasmática , Humanos , Organelas/fisiologia , Plantas/metabolismo , RNA/metabolismo , Ribonucleoproteínas/metabolismo , Vesículas Secretórias/fisiologia , Vesículas Transportadoras/fisiologia
18.
Nat Cell Biol ; 20(3): 344-351, 2018 03.
Artigo em Inglês | MEDLINE | ID: mdl-29403036

RESUMO

Recent advances in cell biology enable precise molecular perturbations. The spatiotemporal organization of cells and organisms, however, also depends on physical processes such as diffusion or cytoplasmic flows, and strategies to perturb physical transport inside cells are not yet available. Here, we demonstrate focused-light-induced cytoplasmic streaming (FLUCS). FLUCS is local, directional, dynamic, probe-free, physiological, and is even applicable through rigid egg shells or cell walls. We explain FLUCS via time-dependent modelling of thermoviscous flows. Using FLUCS, we demonstrate that cytoplasmic flows drive partitioning-defective protein (PAR) polarization in Caenorhabditis elegans zygotes, and that cortical flows are sufficient to transport PAR domains and invert PAR polarity. In addition, we find that asymmetric cell division is a binary decision based on gradually varying PAR polarization states. Furthermore, the use of FLUCS for active microrheology revealed a metabolically induced fluid-to-solid transition of the yeast cytoplasm. Our findings establish how a wide range of transport-dependent models of cellular organization become testable by FLUCS.


Assuntos
Caenorhabditis elegans/fisiologia , Corrente Citoplasmática , Análise de Célula Única/métodos , Zigoto/fisiologia , Animais , Animais Geneticamente Modificados , Caenorhabditis elegans/embriologia , Caenorhabditis elegans/genética , Caenorhabditis elegans/efeitos da radiação , Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismo , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Polaridade Celular , Corrente Citoplasmática/efeitos da radiação , Raios Infravermelhos , Lasers , Modelos Biológicos , Fenótipo , Reologia , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/fisiologia , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Análise de Célula Única/instrumentação , Fatores de Tempo , Zigoto/citologia , Zigoto/metabolismo , Zigoto/efeitos da radiação
19.
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
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