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1.
Int J Mol Sci ; 22(15)2021 Jul 23.
Artigo em Inglês | MEDLINE | ID: mdl-34360622

RESUMO

Kinesin-5 motor consists of two pairs of heads and tail domains, which are situated at the opposite ends of a common stalk. The two pairs of heads can bind to two antiparallel microtubules (MTs) and move on the two MTs independently towards the plus ends, sliding apart the two MTs, which is responsible for chromosome segregation during mitosis. Prior experimental data showed that the tails of kinesin-5 Eg5 can modulate the dynamics of single motors and are critical for multiple motors to generate high steady forces to slide apart two antiparallel MTs. To understand the molecular mechanism of the tails modulating the ability of Eg5 motors, based on our proposed model the dynamics of the single Eg5 with the tails and that without the tails moving on single MTs is studied analytically and compared. Furthermore, the dynamics of antiparallel MT sliding by multiple Eg5 motors with the tails and that without the tails is studied numerically and compared. Both the analytical results for single motors and the numerical results for multiple motors are consistent with the available experimental data.


Assuntos
Cinesina/fisiologia , Microtúbulos/fisiologia , Modelos Moleculares
2.
Nat Commun ; 12(1): 4470, 2021 07 22.
Artigo em Inglês | MEDLINE | ID: mdl-34294690

RESUMO

Gravity is a critical environmental factor regulating directional growth and morphogenesis in plants, and gravitropism is the process by which plants perceive and respond to the gravity vector. The cytoskeleton is proposed to play important roles in gravitropism, but the underlying mechanisms are obscure. Here we use genetic screening in Physcomitrella patens, to identify a locus GTRC, that when mutated, reverses the direction of protonemal gravitropism. GTRC encodes a processive minus-end-directed KCHb kinesin, and its N-terminal, C-terminal and motor domains are all essential for transducing the gravity signal. Chimeric analysis between GTRC/KCHb and KCHa reveal a unique role for the N-terminus of GTRC in gravitropism. Further study shows that gravity-triggered normal asymmetric distribution of actin filaments in the tip of protonema is dependent on GTRC. Thus, our work identifies a microtubule-based cellular motor that determines the direction of plant gravitropism via mediating the asymmetric distribution of actin filaments.


Assuntos
Bryopsida/fisiologia , Gravitropismo/fisiologia , Cinesina/fisiologia , Proteínas de Plantas/fisiologia , Citoesqueleto de Actina/química , Citoesqueleto de Actina/fisiologia , Sequência de Bases , Bryopsida/genética , Mapeamento Cromossômico , Citoesqueleto/química , Citoesqueleto/fisiologia , DNA de Plantas/genética , Genes de Plantas , Gravitropismo/genética , Cinesina/química , Cinesina/genética , Microtúbulos/química , Microtúbulos/fisiologia , Mutagênese , Mutação , Proteínas de Plantas/química , Proteínas de Plantas/genética , Plantas Geneticamente Modificadas , Domínios Proteicos
3.
Gene ; 798: 145795, 2021 Sep 25.
Artigo em Inglês | MEDLINE | ID: mdl-34175396

RESUMO

Spermiogenesis is the final phase of spermatogenesis, wherein the spermatids differentiate into mature spermatozoa via complex morphological transformation. In this process, kinesin plays an important role. Here, we observed the morphological transformation of spermatids and analyzed the characterization, dynamic transcription, and potential function of kinesin KIF3A/KIF3B during spermiogenesis in Chinese hook snout carp (Opsariichthys bidens). We found that the full-length cDNAs of O. bidens kif3a and kif3b were 2544 and 2806 bp in length comprising 119 bp and 259 bp 5' untranslated region (UTR), 313 bp and 222 bp 3' UTR, and 2112 bp and 2325 bp open reading frame encoding 703 and 774 amino acids, respectively. Ob-KIF3A/KIF3B proteins have three domains, namely N-terminal head, coiled-coil stalk, and C-terminal tail, and exhibit high similarity with homologous proteins in vertebrates and invertebrates. Ob-kif3a/kif3b mRNAs were ubiquitously expressed in all tissues examined, with the highest expression in the brain and stage-IV testis. Immunofluorescence results showed that Ob-KIF3A was co-localized with tubulin and the mitochondria. Particularly, in early spermatids, Ob-KIF3A, tubulin, and the mitochondrial signals were evenly distributed in the cytoplasm, whereas in middle spermatids, they were distributed around the nucleus. In the late stage, the signals were concentrated on one side of the nucleus, where the tail is formed, whereas in mature sperms, they were detected in the midpiece and flagellum. These results indicate that Ob-KIF3A/KIF3B may participate in nuclear reshaping, flagellum formation, and mitochondrial aggregation in the midpiece during spermiogenesis.


Assuntos
Cyprinidae/fisiologia , Cinesina/fisiologia , Espermatogênese/fisiologia , Animais , Cyprinidae/genética , Cinesina/química , Cinesina/genética , Masculino , Microtúbulos/metabolismo , Mitocôndrias/metabolismo , Filogenia , Conformação Proteica , RNA Mensageiro/metabolismo , Alinhamento de Sequência , Análise de Sequência de DNA , Cauda do Espermatozoide/fisiologia , Espermátides/fisiologia , Espermátides/ultraestrutura , Espermatogênese/genética , Testículo/metabolismo , Transcrição Genética
4.
Dev Biol ; 477: 191-204, 2021 09.
Artigo em Inglês | MEDLINE | ID: mdl-34090925

RESUMO

Development of sperm requires microtubule-based movements that drive assembly of a compact head and flagellated tails. Much is known about how flagella are built given their shared molecular core with motile cilia, but less is known about the mechanisms that shape the sperm head. The Kinesin Superfamily Protein 3A (KIF3A) pairs off with a second motor protein (KIF3B) and the Kinesin Associated Protein 3 (KAP3) to form Heterotrimeric Kinesin II. This complex drives intraflagellar transport (IFT) along microtubules during ciliary assembly. We show that KIF3A and KAP3 orthologs in Schmidtea mediterranea are required for axonemal assembly and nuclear elongation during spermiogenesis. Expression of Smed-KAP3 is enriched during planarian spermatogenesis with transcript abundance peaking in spermatocyte and spermatid cells. Disruption of Smed-kif3A or Smed-KAP3 expression by RNA-interference results in loss of spermatozoa and accumulation of unelongated spermatids. Confocal microscopy of planarian testis lobes stained with alpha-tubulin antibodies revealed that spermatids with disrupted Kinesin II function fail to assemble flagella, and visualization with 4',6-diamidino-2-phenylindole (DAPI) revealed reduced nuclear elongation. Disruption of Smed-kif3A or Smed-KAP3 expression also resulted in edema, reduced locomotion, and loss of epidermal cilia, which corroborates with somatic phenotypes previously reported for Smed-kif3B. These findings demonstrate that heterotrimeric Kinesin II drives assembly of cilia and flagella, as well as rearrangements of nuclear morphology in developing sperm. Prolonged activity of heterotrimeric Kinesin II in manchette-like structures with extended presence during spermiogenesis is hypothesized to result in the exaggerated nuclear elongation observed in sperm of turbellarians and other lophotrochozoans.


Assuntos
Cinesina/fisiologia , Planárias/citologia , Cauda do Espermatozoide/fisiologia , Espermatogênese/fisiologia , Animais , Núcleo Celular/ultraestrutura , Proteínas do Citoesqueleto/fisiologia , Técnicas de Silenciamento de Genes , Cinesina/química , Cinesina/genética , Masculino , Interferência de RNA , Cabeça do Espermatozoide/ultraestrutura , Cauda do Espermatozoide/ultraestrutura
5.
Commun Biol ; 4(1): 552, 2021 05 11.
Artigo em Inglês | MEDLINE | ID: mdl-33976373

RESUMO

During mammalian brain development, neural progenitor cells proliferate extensively but can ensure the production of correct numbers of various types of mature cells by balancing symmetric proliferative versus asymmetric differentiative cell divisions. This process of cell fate determination may be harnessed for developing cancer therapy. Here, we test this idea by targeting KIF20A, a mitotic kinesin crucial for the control of cell division modes, in a genetic model of medulloblastoma (MB) and human MB cells. Inducible Kif20a knockout in both normal and MB-initiating granule neuron progenitors (GNPs) causes early cell cycle exit and precocious neuronal differentiation without causing cytokinesis failure and suppresses the development of Sonic Hedgehog (SHH)-activated MB. Inducible KIF20A knockdown in human MB cells inhibits proliferation both in cultures and in growing tumors. Our results indicate that targeting the fate specification process of nascent daughter cells presents a novel avenue for developing anti-proliferation treatment for malignant brain tumors.


Assuntos
Cinesina/metabolismo , Meduloblastoma/metabolismo , Células-Tronco Neurais/metabolismo , Animais , Ciclo Celular/genética , Diferenciação Celular/fisiologia , Proliferação de Células/fisiologia , Neoplasias Cerebelares/genética , Neoplasias Cerebelares/patologia , Proteínas Hedgehog/metabolismo , Cinesina/genética , Cinesina/fisiologia , Meduloblastoma/fisiopatologia , Camundongos , Camundongos Knockout , Células-Tronco Neurais/fisiologia , Neurônios/metabolismo , Transdução de Sinais/fisiologia , Células-Tronco/metabolismo
6.
Cancer Lett ; 506: 1-10, 2021 05 28.
Artigo em Inglês | MEDLINE | ID: mdl-33652084

RESUMO

Treatment of aggressive meningiomas remains challenging due to a high rate of recurrence in higher-grade meningiomas, frequent subtotal resections, and the lack of effective systemic treatments. Substantial overexpression associated with a poor prognosis has been demonstrated for kinesin family member 11 (KIF11) in high-grade meningiomas. Due to anti-tumor activity for KIF11 inhibitors (KIF11i) filanesib and ispinesib in other cancer types, we sought to investigate their mode of action and efficacy for the treatment of aggressive meningiomas. Dose curve analysis of both KIF11i revealed IC50 values of less than 1 nM in anaplastic and benign meningioma cell lines. Both compounds induced G2/M arrest and subsequent subG1 increase in all cell lines. Profound induction of apoptosis was detected in the anaplastic cell lines determined by annexin V staining. KIF11i significantly inhibited meningioma growth in xenotransplanted mice by up to 83%. Furthermore, both drugs induced minor hematological side effects, which were less pronounced for filanesib. We identified substantial in vitro and in vivo anti-tumor effects of the KIF11 inhibitors filanesib and ispinesib, with filanesib demonstrating better tolerability, suggesting future use of filanesib for the treatment of aggressive meningioma.


Assuntos
Benzamidas/farmacologia , Cinesina/antagonistas & inibidores , Neoplasias Meníngeas/tratamento farmacológico , Meningioma/tratamento farmacológico , Quinazolinas/farmacologia , Tiadiazóis/farmacologia , Animais , Benzamidas/uso terapêutico , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Feminino , Pontos de Checagem da Fase G2 do Ciclo Celular/efeitos dos fármacos , Humanos , Cinesina/fisiologia , Neoplasias Meníngeas/patologia , Meningioma/patologia , Camundongos , Quinazolinas/uso terapêutico , Tiadiazóis/uso terapêutico , Ensaios Antitumorais Modelo de Xenoenxerto
7.
Mol Biol Cell ; 32(7): 590-604, 2021 04 01.
Artigo em Inglês | MEDLINE | ID: mdl-33566676

RESUMO

The asymmetric distribution of microtubule (MT) dynamics in migrating cells is important for cell polarization, yet the underlying regulatory mechanisms remain underexplored. Here, we addressed this question by studying the role of the MT depolymerase, MCAK (mitotic centromere-associated kinesin), in the highly persistent migration of RPE-1 cells. MCAK knockdown leads to slowed migration and poor directional movement. Fixed and live cell imaging revealed that MCAK knockdown results in excessive membrane ruffling as well as defects in cell polarization and the maintenance of a major protrusive front. Additionally, loss of MCAK increases the lifetime of focal adhesions by decreasing their disassembly rate. These functions correlate with a spatial distribution of MCAK activity, wherein activity is higher in the trailing edge of cells compared with the leading edge. Overexpression of Rac1 has a dominant effect over MCAK activity, placing it downstream of or in a parallel pathway to MCAK function in migration. Together, our data support a model in which the polarized distribution of MCAK activity and subsequent differential regulation of MT dynamics contribute to cell polarity, centrosome positioning, and focal adhesion dynamics, which all help facilitate robust directional migration.


Assuntos
Polaridade Celular/fisiologia , Adesões Focais/metabolismo , Cinesina/metabolismo , Adesão Celular/fisiologia , Movimento Celular/fisiologia , Centrômero/metabolismo , Humanos , Cinesina/fisiologia , Microtúbulos/metabolismo , Microtúbulos/fisiologia , Mitose , Fosforilação , Proteínas Serina-Treonina Quinases/metabolismo
8.
Mol Biol Cell ; 32(9): 984-994, 2021 04 19.
Artigo em Inglês | MEDLINE | ID: mdl-33439674

RESUMO

Cellular cargoes, including lipid droplets and mitochondria, are transported along microtubules using molecular motors such as kinesins. Many experimental and computational studies focused on cargoes with rigidly attached motors, in contrast to many biological cargoes that have lipid surfaces that may allow surface mobility of motors. We extend a mechanochemical three-dimensional computational model by adding coupled-viscosity effects to compare different motor arrangements and mobilities. We show that organizational changes can optimize for different objectives: Cargoes with clustered motors are transported efficiently but are slow to bind to microtubules, whereas those with motors dispersed rigidly on their surface bind microtubules quickly but are transported inefficiently. Finally, cargoes with freely diffusing motors have both fast binding and efficient transport, although less efficient than clustered motors. These results suggest that experimentally observed changes in motor organization may be a control point for transport.


Assuntos
Biologia Computacional/métodos , Cinesina/metabolismo , Transporte Proteico/fisiologia , Transporte Biológico , Fenômenos Biofísicos , Difusão , Cinesina/fisiologia , Microtúbulos/metabolismo , Modelos Teóricos , Proteínas Motores Moleculares/metabolismo , Viscosidade
9.
Cancer Res ; 81(4): 1026-1039, 2021 02 15.
Artigo em Inglês | MEDLINE | ID: mdl-33277366

RESUMO

The new generation androgen receptor (AR) pathway inhibitor enzalutamide can prolong the survival of patients with metastatic prostate cancer. However, resistance to enzalutamide inevitably develops in these patients, and the underlying mechanisms of this resistance are not fully defined. Here we demonstrate that the kinesin family member 15 (KIF15) contributes to enzalutamide resistance by enhancing the AR signaling in prostate cancer cells. KIF15 directly bound the N-terminus of AR/AR-V7 and prevented AR/AR-V7 proteins from degradation by increasing the protein association of ubiquitin-specific protease 14 (USP14) with AR/AR-V7. In turn, the transcriptionally active AR stimulated KIF15 expression. KIF15 inhibitors alone or in combination with enzalutamide significantly suppressed enzalutamide-resistant prostate cancer cell growth and xenograft progression. These findings highlight a key role of KIF15 in enabling prostate cancer cells to develop therapy resistance to enzalutamide and rationalize KIF15 as a potential therapeutic target. SIGNIFICANCE: These findings demonstrate how reciprocal activation between KIF15 and AR contributes to enzalutamide resistance in prostate cancer and highlights cotargeting KIF15 and AR as a therapeutic strategy for these tumors.


Assuntos
Benzamidas/uso terapêutico , Resistencia a Medicamentos Antineoplásicos/genética , Cinesina/fisiologia , Nitrilas/uso terapêutico , Feniltioidantoína/uso terapêutico , Neoplasias da Próstata/tratamento farmacológico , Receptores Androgênicos/metabolismo , Animais , Linhagem Celular Tumoral , Progressão da Doença , Células HEK293 , Humanos , Cinesina/genética , Masculino , Camundongos , Camundongos Nus , Células PC-3 , Neoplasias da Próstata/genética , Neoplasias da Próstata/metabolismo , Neoplasias da Próstata/patologia , Domínios Proteicos/genética , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Estabilidade Proteica , Proteólise , Receptores Androgênicos/química , Receptores Androgênicos/genética
10.
Dev Biol ; 469: 26-36, 2021 01 01.
Artigo em Inglês | MEDLINE | ID: mdl-32961118

RESUMO

Kinesins are microtubule-based motor proteins that are well known for their key roles in cell biological processes ranging from cell division, to intracellular transport of mRNAs, proteins, vesicles, and organelles, and microtubule disassembly. Interestingly, many of the ~45 distinct kinesin genes in vertebrate genomes have also been associated with specific phenotypes in embryonic development. In this review, we highlight the specific developmental roles of kinesins, link these to cellular roles reported in vitro, and highlight remaining gaps in our understanding of how this large and important family of proteins contributes to the development and morphogenesis of animals.


Assuntos
Desenvolvimento Embrionário , Cinesina/fisiologia , Animais , Transporte Biológico , Ciclo Celular , Sistema Nervoso Central/embriologia , Cílios/fisiologia , Doenças Genéticas Inatas/etiologia , Humanos , Cinesina/química , Mitose , Organogênese
11.
J Neurosci ; 40(48): 9169-9185, 2020 11 25.
Artigo em Inglês | MEDLINE | ID: mdl-33097641

RESUMO

Myosin X (Myo X) transports cargos to the tips of filopodia for cell adhesion, migration, and neuronal axon guidance. Deleted in Colorectal Cancer (DCC) is one of the Myo X cargos that is essential for Netrin-1-regulated axon pathfinding. The function of Myo X in axon development in vivo and the underlying mechanisms remain elusive. Here, we provide evidence for the role of Myo X in Netrin-1-DCC-regulated axon development in developing mouse neocortex. The knockout (KO) or knockdown (KD) of Myo X in cortical neurons of embryonic mouse brain impairs axon initiation and contralateral branching/targeting. Similar axon deficits are detected in Netrin-1-KO or DCC-KD cortical neurons. Further proteomic analysis of Myo X binding proteins identifies KIF13B (a kinesin family motor protein). The Myo X interaction with KIF13B is induced by Netrin-1. Netrin-1 promotes anterograde transportation of Myo X into axons in a KIF13B-dependent manner. KIF13B-KD cortical neurons exhibit similar axon deficits. Together, these results reveal Myo X-KIF13B as a critical pathway for Netrin-1-promoted axon initiation and branching/targeting.SIGNIFICANCE STATEMENT Netrin-1 increases Myosin X (Myo X) interaction with KIF13B, and thus promotes axonal delivery of Myo X and axon initiation and contralateral branching in developing cerebral neurons, revealing unrecognized functions and mechanisms underlying Netrin-1 regulation of axon development.


Assuntos
Axônios/fisiologia , Cinesina/fisiologia , Proteínas de Membrana/fisiologia , Miosinas/fisiologia , Netrina-1/fisiologia , Animais , Células Cultivadas , Córtex Cerebral/citologia , Córtex Cerebral/crescimento & desenvolvimento , Receptor DCC/genética , Receptor DCC/fisiologia , Feminino , Cinesina/genética , Masculino , Proteínas de Membrana/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Miosinas/genética , Neocórtex/citologia , Neocórtex/crescimento & desenvolvimento , Netrina-1/genética , Gravidez
12.
Elife ; 92020 10 28.
Artigo em Inglês | MEDLINE | ID: mdl-33112235

RESUMO

Heterodimeric motor organization of kinesin-II is essential for its function in anterograde IFT in ciliogenesis. However, the underlying mechanism is not well understood. In addition, the anterograde IFT velocity varies significantly in different organisms, but how this velocity affects ciliary length is not clear. We show that in Chlamydomonas motors are only stable as heterodimers in vivo, which is likely the key factor for the requirement of a heterodimer for IFT. Second, chimeric CrKinesin-II with human kinesin-II motor domains functioned in vitro and in vivo, leading to a ~ 2.8 fold reduced anterograde IFT velocity and a similar fold reduction in IFT injection rate that supposedly correlates with ciliary assembly activity. However, the ciliary length was only mildly reduced (~15%). Modeling analysis suggests a nonlinear scaling relationship between IFT velocity and ciliary length that can be accounted for by limitation of the motors and/or its ciliary cargoes, e.g. tubulin.


Assuntos
Proteínas de Algas/metabolismo , Chlamydomonas reinhardtii/fisiologia , Cílios/fisiologia , Cinesina/metabolismo , Proteínas de Algas/fisiologia , Chlamydomonas reinhardtii/metabolismo , Cílios/metabolismo , Cinesina/fisiologia
13.
J Neurogenet ; 34(3-4): 282-297, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-33030066

RESUMO

Axonal transport is integral for maintaining neuronal form and function, and defects in axonal transport have been correlated with several neurological diseases, making it a subject of extensive research over the past several years. The anterograde and retrograde transport machineries are crucial for the delivery and distribution of several cytoskeletal elements, growth factors, organelles and other synaptic cargo. Molecular motors and the neuronal cytoskeleton function as effectors for multiple neuronal processes such as axon outgrowth and synapse formation. This review examines the molecular mechanisms governing axonal transport, specifically highlighting the contribution of studies conducted in C. elegans, which has proved to be a tractable model system in which to identify both novel and conserved regulatory mechanisms of axonal transport.


Assuntos
Transporte Axonal/fisiologia , Proteínas de Caenorhabditis elegans/fisiologia , Caenorhabditis elegans/fisiologia , Proteínas do Tecido Nervoso/fisiologia , Actinas/fisiologia , Animais , Caenorhabditis elegans/citologia , Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/genética , Citoesqueleto/fisiologia , Proteínas de Filamentos Intermediários/fisiologia , Cinesina/fisiologia , Microtúbulos/fisiologia , Proteínas Motores Moleculares/fisiologia , Proteínas do Tecido Nervoso/genética , Neurônios/citologia , Neurônios/fisiologia , Organelas , Processamento de Proteína Pós-Traducional , Vesículas Sinápticas
14.
Sci Rep ; 10(1): 13864, 2020 08 17.
Artigo em Inglês | MEDLINE | ID: mdl-32807823

RESUMO

Armadillo (Arm) is crucial for transducing Wingless (Wg) signaling. Previously, we have shown that Klp64D, a motor subunit of Drosophila kinesin-II, interacts with Arm for Wg signaling. Molecular basis for this interaction has remained unknown. Here we identify a critical Arm repeat (AR) required for binding Klp64D and Wg signaling. Arm/[Formula: see text]-catenin family proteins contain a conserved domain of 12 Arm repeats (ARs). Five of these ARs can interact with Klp64D, but only the second AR (AR2) binds to the cargo/tail domain of Klp64D. Overexpression of AR2 in wing imaginal disc is sufficient to cause notched wing margin. This phenotype by AR2 is enhanced or suppressed by reducing or increasing Klp64D expression, respectively. AR2 overexpression inhibits Wg signaling activity in TopFlash assay, consistent with its dominant-negative effects on Klp64D-dependent Wg signaling. Overexpression of the Klp64D cargo domain also results in dominant-negative wing notching. Genetic rescue data indicate that both AR2 and Klp64D cargo regions are required for the function of Arm and Klp64D, respectively. AR2 overexpression leads to an accumulation of Arm with GM130 Golgi marker in Klp64D knockdown. This study suggests that Wg signaling for wing development is regulated by specific interaction between AR2 and the cargo domain of Klp64D.


Assuntos
Proteínas do Domínio Armadillo/genética , Proteínas do Domínio Armadillo/fisiologia , Proteínas de Drosophila/genética , Proteínas de Drosophila/fisiologia , Drosophila/crescimento & desenvolvimento , Regulação da Expressão Gênica no Desenvolvimento , Cinesina/genética , Fatores de Transcrição/genética , Fatores de Transcrição/fisiologia , Asas de Animais/crescimento & desenvolvimento , Proteína Wnt1/genética , Animais , Drosophila/genética , Genes de Insetos , Cinesina/fisiologia , Via de Sinalização Wnt
15.
Methods Mol Biol ; 2143: 271-292, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32524487

RESUMO

Axonal transport, which is the process mediating the active shuttling of a variety cargoes from one end of an axon to the other, is essential for the development, function, and survival of neurons. Impairments in this dynamic process are linked to diverse nervous system diseases and advanced ageing. It is thus essential that we quantitatively study the kinetics of axonal transport to gain an improved understanding of neuropathology as well as the molecular and cellular mechanisms regulating cargo trafficking. One of the best ways to achieve this goal is by imaging individual, fluorescent cargoes in live systems and analyzing the kinetic properties of their progression along the axon. We have therefore developed an intravital technique to visualize different organelles, such as signaling endosomes and mitochondria, being actively transported in the axons of both motor and sensory neurons in live, anesthetized rodents. In this chapter, we provide step-by-step instructions on how to deliver specific organelle-targeting, fluorescent probes using several routes of administration to image individual cargoes being bidirectionally transported along axons within the exposed sciatic nerve. This method can provide detailed, physiologically relevant information on axonal transport, and is thus poised to elucidate mechanisms regulating this process in both health and disease.


Assuntos
Transporte Axonal/fisiologia , Microscopia Intravital/métodos , Degeneração Neural/patologia , Nervos Periféricos/fisiologia , Animais , Endossomos/ultraestrutura , Corantes Fluorescentes/administração & dosagem , Corantes Fluorescentes/análise , Corantes Fluorescentes/farmacocinética , Genes Reporter , Injeções Intramusculares , Microscopia Intravital/instrumentação , Cinesina/fisiologia , Músculo Esquelético , Organelas/ultraestrutura , Nervos Periféricos/ultraestrutura , Roedores , Nervo Isquiático/fisiologia , Nervo Isquiático/ultraestrutura
16.
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
Cinesina/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 , Cinesina/fisiologia , Fenômenos Mecânicos , Microtúbulos/metabolismo , Fluxo Óptico , Orientação Espacial/fisiologia
17.
Mol Biol Cell ; 31(16): 1802-1814, 2020 07 21.
Artigo em Inglês | MEDLINE | ID: mdl-32129712

RESUMO

How cells position their organelles is a fundamental biological question. During Drosophila embryonic muscle development, multiple nuclei transition from being clustered together to splitting into two smaller clusters to spreading along the myotube's length. Perturbations of microtubules and motor proteins disrupt this sequence of events. These perturbations do not allow intuiting which molecular forces govern the nuclear positioning; we therefore used computational screening to reverse-engineer and identify these forces. The screen reveals three models. Two suggest that the initial clustering is due to nuclear repulsion from the cell poles, while the third, most robust, model poses that this clustering is due to a short-ranged internuclear attraction. All three models suggest that the nuclear spreading is due to long-ranged internuclear repulsion. We test the robust model quantitatively by comparing it with data from perturbed muscle cells. We also test the model using agent-based simulations with elastic dynamic microtubules and molecular motors. The model predicts that, in longer mammalian myotubes with a large number of nuclei, the spreading stage would be preceded by segregation of the nuclei into a large number of clusters, proportional to the myotube length, with a small average number of nuclei per cluster.


Assuntos
Núcleo Celular/fisiologia , Drosophila melanogaster/embriologia , Microtúbulos/metabolismo , Animais , Transporte Biológico , Núcleo Celular/metabolismo , Análise por Conglomerados , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/metabolismo , Dineínas/metabolismo , Dineínas/fisiologia , Cinesina/metabolismo , Cinesina/fisiologia , Microtúbulos/fisiologia , Modelos Biológicos , Desenvolvimento Muscular/fisiologia , Fibras Musculares Esqueléticas/metabolismo , Miosinas/metabolismo
18.
Curr Biol ; 30(6): 1160-1166.e5, 2020 03 23.
Artigo em Inglês | MEDLINE | ID: mdl-32142698

RESUMO

Construction and function of virtually all cilia require the universally conserved process of intraflagellar transport (IFT) [1, 2]. During the atypically fast IFT in the green alga C. reinhardtii, on average, 10 kinesin-2 motors "line up" in a tight assembly on the trains [3], provoking the question of how these motors coordinate their action to ensure smooth and fast transport along the flagellum without standing in each other's way. Here, we show that the heterodimeric FLA8/10 kinesin-2 alone is responsible for the atypically fast IFT in C. reinhardtii. Notably, in single-molecule studies, FLA8/10 moved at speeds matching those of in vivo IFT [4] but additionally displayed a slow velocity distribution, indicative of auto-inhibition. Addition of the KAP subunit to generate the heterotrimeric FLA8/10/KAP relieved this inhibition, thus providing a mechanistic rationale for heterotrimerization with the KAP subunit fully activating FLA8/10 for IFT in vivo. Finally, we linked fast FLA8/10 and slow KLP11/20 kinesin-2 from C. reinhardtii and C. elegans through a DNA tether to understand the molecular underpinnings of motor coordination during IFT in vivo. For motor pairs from both species, the co-transport velocities very nearly matched the single-molecule velocities, and both complexes spent roughly 80% of the time with only one of the two motors attached to the microtubule. Thus, irrespective of phylogeny and kinetic properties, kinesin-2 motors work mostly alone without sacrificing efficiency. Our findings thus offer a simple mechanism for how efficient IFT is achieved across diverse organisms despite being carried out by motors with different properties.


Assuntos
Chlamydomonas reinhardtii/fisiologia , Cinesina/fisiologia , Proteínas Associadas aos Microtúbulos/genética , Proteínas de Protozoários/genética , Transporte Biológico , Chlamydomonas reinhardtii/genética , Flagelos/fisiologia , Proteínas Associadas aos Microtúbulos/metabolismo , Transporte Proteico , Proteínas de Protozoários/metabolismo
19.
J Neurochem ; 155(1): 10-28, 2020 10.
Artigo em Inglês | MEDLINE | ID: mdl-32196676

RESUMO

One of the characteristic features of different classes of neurons that is vital for their proper functioning within neuronal networks is the shape of their dendritic arbors. To properly develop dendritic trees, neurons need to accurately control the intracellular transport of various cellular cargo (e.g., mRNA, proteins, and organelles). Microtubules and motor proteins (e.g., dynein and kinesins) that move along microtubule tracks play an essential role in cargo sorting and transport to the most distal ends of neurons. Equally important are motor adaptors, which may affect motor activity and specify cargo that is transported by the motor. Such transport undergoes very dynamic fine-tuning in response to changes in the extracellular environment and synaptic transmission. Such regulation is achieved by the phosphorylation of motors, motor adaptors, and cargo, among other mechanisms. This review focuses on the contribution of the dynein-dynactin complex, kinesins, their adaptors, and the phosphorylation of these proteins in the formation of dendritic trees by maturing neurons. We primarily review the effects of the motor activity of these proteins in dendrites on dendritogenesis. We also discuss less anticipated mechanisms that contribute to dendrite growth, such as dynein-driven axonal transport and non-motor functions of kinesins.


Assuntos
Dendritos , Complexo Dinactina/fisiologia , Dineínas/fisiologia , Cinesina/fisiologia , Neurônios Motores/fisiologia , Animais , Humanos , Neurogênese/fisiologia , Fosforilação
20.
J Virol ; 94(9)2020 04 16.
Artigo em Inglês | MEDLINE | ID: mdl-32075931

RESUMO

Alphaherpesviruses, including pseudorabies virus (PRV), are neuroinvasive pathogens that establish lifelong latency in peripheral ganglia following the initial infection at mucosal surfaces. The establishment of latent infection and subsequent reactivations, during which newly assembled virions are sorted into and transported anterogradely inside axons to the initial mucosal site of infection, rely on axonal bidirectional transport mediated by microtubule-based motors. Previous studies using cultured peripheral nervous system (PNS) neurons have demonstrated that KIF1A, a kinesin-3 motor, mediates the efficient axonal sorting and transport of newly assembled PRV virions. Here we report that KIF1A, unlike other axonal kinesins, is an intrinsically unstable protein prone to proteasomal degradation. Interestingly, PRV infection of neuronal cells leads not only to a nonspecific depletion of KIF1A mRNA but also to an accelerated proteasomal degradation of KIF1A proteins, leading to a near depletion of KIF1A protein late in infection. Using a series of PRV mutants deficient in axonal sorting and anterograde spread, we identified the PRV US9/gE/gI protein complex as a viral factor facilitating the proteasomal degradation of KIF1A proteins. Moreover, by using compartmented neuronal cultures that fluidically and physically separate axons from cell bodies, we found that the proteasomal degradation of KIF1A occurs in axons during infection. We propose that the PRV anterograde sorting complex, gE/gI/US9, recruits KIF1A to viral transport vesicles for axonal sorting and transport and eventually accelerates the proteasomal degradation of KIF1A in axons.IMPORTANCE Pseudorabies virus (PRV) is an alphaherpesvirus related to human pathogens herpes simplex viruses 1 and 2 and varicella-zoster virus. Alphaherpesviruses are neuroinvasive pathogens that establish lifelong latent infections in the host peripheral nervous system (PNS). Following reactivation from latency, infection spreads from the PNS back via axons to the peripheral mucosal tissues, a process mediated by kinesin motors. Here, we unveil and characterize the underlying mechanisms for a PRV-induced, accelerated degradation of KIF1A, a kinesin-3 motor promoting the sorting and transport of PRV virions in axons. We show that PRV infection disrupts the synthesis of KIF1A and simultaneously promotes the degradation of intrinsically unstable KIF1A proteins by proteasomes in axons. Our work implies that the timing of motor reduction after reactivation would be critical because progeny particles would have a limited time window for sorting into and transport in axons for further host-to-host spread.


Assuntos
Herpesvirus Suídeo 1/metabolismo , Cinesina/metabolismo , Pseudorraiva/metabolismo , Animais , Transporte Axonal/fisiologia , Axônios/virologia , Linhagem Celular , Células Cultivadas , Interações Hospedeiro-Patógeno , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Cinesina/fisiologia , Masculino , Microtúbulos/metabolismo , Neurônios/virologia , Cultura Primária de Células , Ratos , Ratos Sprague-Dawley , Proteínas do Envelope Viral/genética , Vírion/metabolismo
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