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1.
Results Probl Cell Differ ; 67: 27-48, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31435791

RESUMO

Formin homology proteins (formins) are a highly conserved family of cytoskeletal remodeling proteins that are involved in a diverse array of cellular functions. Formins are best known for their ability to regulate actin dynamics, but the same functional domains also govern stability and organization of microtubules. It is thought that this dual activity allows them to coordinate the activity of these two major cytoskeletal networks and thereby influence cellular architecture. Golgi ribbon assembly is dependent upon cooperative interactions between actin filaments and cytoplasmic microtubules originating both at the Golgi itself and from the centrosome. Similarly, centrosome assembly, centriole duplication, and centrosome positioning are also reliant on a dialogue between both cytoskeletal networks. As presented in this chapter, a growing body of evidence suggests that multiple formin proteins play essential roles in these central cellular processes.


Assuntos
Centríolos/metabolismo , Proteínas do Citoesqueleto/metabolismo , Citoesqueleto/metabolismo , Complexo de Golgi/metabolismo , Citoesqueleto de Actina/metabolismo , Actinas/metabolismo , Microtúbulos/metabolismo
2.
Results Probl Cell Differ ; 67: 323-336, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31435801

RESUMO

The centrosome, a major microtubule organizer, has important functions in regulating the cytoskeleton as well as the position of cellular structures and orientation of cells within tissues. The centrosome serves as the main cytoskeleton-organizing centre in the cell and is the classical site of microtubule nucleation and anchoring. For these reasons, centrosomes play a very important role in morphogenesis, not just in the early stages of cell divisions but also in the later stages of organogenesis. Many organs such as lung, kidney and blood vessels develop from epithelial tubes that branch into complex networks. Cells in the nervous system also form highly branched structures in order to build complex neuronal networks. During branching morphogenesis, cells have to rearrange within tissues though multicellular branching or through subcellular branching, also known as single-cell branching. For highly branched structures to be formed during embryonic development, the cytoskeleton needs to be extensively remodelled. The centrosome has been shown to play an important role during these events.


Assuntos
Centrossomo/fisiologia , Desenvolvimento Embrionário , Morfogênese , Microtúbulos/metabolismo , Organogênese
3.
Gene ; 719: 144074, 2019 Nov 30.
Artigo em Inglês | MEDLINE | ID: mdl-31446094

RESUMO

Kinesin-14 motor es-kifc1 is highly expressed in the male reproductive system of the Chinese mitten crab Eriocheir sinensis (E. sinensis). In addition to acrosomal formation, es-KIFC1 also tightly surrounds the nucleus and its specific mechanism remains unknown. During spermatogenesis, sperm nucleus dents into a cup-shaped structure with several radial arms and completed the nuclear decondensation. In this study, the spatial expression pattern of es-KIFC1 indicates a potential function in nuclear formation with the nuclear localization sequence (NLS) on N-terminal domain which is crucial for the translocation of es-KIFC1 into the nucleus. The Motor domain is associated with microtubule modulation and the Golgi vesicles positioning. Furthermore, the expression level of es-KIFC1 is not only related to the seasonal variation of crustacean development, but also associates with mature sperm storage. The double strand RNA (dsRNA) mediated RNA interference manifests that the cup-shaped sperm nucleus is remarkably malformed and even separates the chromatin throughout the nuclei at the last stage of spermiogenesis. Besides, the sperm nucleus almost disperses its structure and separates the chromatin into several segments throughout the nucleus showing an asymmetrical performance without cytoskeleton. In summary, these results indicate the importance of es-KIFC1 in microtubule positioning and the maintenance of the mature sperm nuclei.


Assuntos
Proteínas de Artrópodes/genética , Braquiúros/fisiologia , Núcleo Celular/metabolismo , Cinesina/genética , Espermatogênese/genética , Animais , Proteínas de Artrópodes/metabolismo , Núcleo Celular/ultraestrutura , Citoesqueleto/metabolismo , Cinesina/metabolismo , Masculino , Microtúbulos/metabolismo , Transporte Proteico , RNA de Cadeia Dupla/genética , Espermatozoides/ultraestrutura
4.
Expert Opin Ther Pat ; 29(8): 623-641, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31353978

RESUMO

Introduction: About 20 patents have been published from 2013 to 2018 for developing advanced cancer therapeutics by targeting tubulin polymerization. Currently, there are several tubulin inhibitors that are in the drug development pipeline for various cancers alone or in combination including antibody-conjugated drugs (ACDs). Areas covered: Important patents focusing on the development of tubulin inhibitors published from 2013 to 2018 are covered. This review mainly focuses on the tubulin inhibitors that are being synthesized and studied in cancer research along with their structures and their phases of development in preclinical and clinical research. Expert opinion: Regulation of microtubules is important for cell division, cell motility, intracellular transport, and cell shape maintenance. Modulating its activity proved to be very effective in various diseases including different types of cancers. Microtubules are composed of two units, namely, alpha and beta-tubulin, and modifications at these ends affect both its functions and dynamics. A number of compounds that have been designed and synthesized bearing various heterocyclic scaffolds have been proven to modulate its activity and have emerged as potent tubulin inhibitors. This encourages more to study microtubules in order to find a variety of novel, potent compounds as anticancer drugs.


Assuntos
Antineoplásicos/farmacologia , Neoplasias/tratamento farmacológico , Moduladores de Tubulina/farmacologia , Animais , Antineoplásicos/química , Desenho de Drogas , Desenvolvimento de Medicamentos/métodos , Humanos , Microtúbulos/efeitos dos fármacos , Microtúbulos/metabolismo , Neoplasias/patologia , Patentes como Assunto , Relação Estrutura-Atividade , Tubulina (Proteína)/efeitos dos fármacos , Tubulina (Proteína)/metabolismo , Moduladores de Tubulina/química
5.
Biochemistry (Mosc) ; 84(4): 358-369, 2019 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-31228927

RESUMO

Cytoplasmic actin structures are essential components of the eukaryotic cytoskeleton. According to the classic concepts, actin structures perform contractile and motor functions, ensuring the possibility of cell shape changes during cell spreading, polarization, and movement both in vitro and in vivo, from the early embryogenesis stages and throughout the life of a multicellular organism. Intracellular organization of actin structures, their biochemical composition, and dynamic properties play a key role in the realization of specific cellular and tissue functions and vary in different cell types. This paper is a review of recent studies on the organization and properties of actin structures in endotheliocytes, interaction of these structures with other cytoskeletal components and elements involved in cell adhesion, as well as their role in the functional activity of endothelial cells.


Assuntos
Citoesqueleto de Actina/metabolismo , Actinas/metabolismo , Citoesqueleto de Actina/química , Actinas/química , Actinas/genética , Caderinas/química , Caderinas/metabolismo , Citosol/metabolismo , Células Endoteliais/citologia , Células Endoteliais/metabolismo , Humanos , Proteínas Associadas aos Microtúbulos/metabolismo , Microtúbulos/química , Microtúbulos/metabolismo
6.
Expert Opin Investig Drugs ; 28(6): 513-523, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-31159588

RESUMO

INTRODUCTION: The management of non-small cell lung cancer (NSCLC) has been substantially improved in the last few years; it has been revolutionized by a patient-tailored approach, especially in the oncogene addicted disease, and by novel combinations containing immune checkpoint inhibitors. However, chemotherapy still represents a mainstay that persists over the decades with limited novelties. Tubulin inhibitors belong to different sub-classes of drugs that share the capability to interfere with mitosis by a direct action on the microtubule system. Among them, taxanes and vinca alkaloids still have a prominent role in clinical practice. AREAS COVERED: This review summarizes the mechanisms of action, current role and future directions of microtubule targeting agents; we focus on investigational agents in phase I and II clinical trials. EXPERT OPINION: Chemotherapy maintains a pivotal role in the treatment of NSCLC. New generation agents that have the potential to overcome the mechanisms of resistance to the available drugs may provide new therapeutic opportunities. Predictive biomarkers derived from combination strategies and phase III studies are necessary going forward.


Assuntos
Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Neoplasias Pulmonares/tratamento farmacológico , Moduladores de Tubulina/administração & dosagem , Animais , Antineoplásicos/administração & dosagem , Antineoplásicos/farmacologia , Protocolos de Quimioterapia Combinada Antineoplásica/administração & dosagem , Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Biomarcadores Tumorais/metabolismo , Carcinoma Pulmonar de Células não Pequenas/patologia , Desenho de Drogas , Drogas em Investigação/administração & dosagem , Drogas em Investigação/farmacologia , Humanos , Neoplasias Pulmonares/patologia , Microtúbulos/efeitos dos fármacos , Microtúbulos/metabolismo , Moduladores de Tubulina/farmacologia
7.
Nat Commun ; 10(1): 2693, 2019 06 19.
Artigo em Inglês | MEDLINE | ID: mdl-31217419

RESUMO

The kinesin-3 KIF1C is a fast organelle transporter implicated in the transport of dense core vesicles in neurons and the delivery of integrins to cell adhesions. Here we report the mechanisms of autoinhibition and release that control the activity of KIF1C. We show that the microtubule binding surface of KIF1C motor domain interacts with its stalk and that these autoinhibitory interactions are released upon binding of protein tyrosine phosphatase PTPN21. The FERM domain of PTPN21 stimulates dense core vesicle transport in primary hippocampal neurons and rescues integrin trafficking in KIF1C-depleted cells. In vitro, human full-length KIF1C is a processive, plus-end directed motor. Its landing rate onto microtubules increases in the presence of either PTPN21 FERM domain or the cargo adapter Hook3 that binds the same region of KIF1C tail. This autoinhibition release mechanism allows cargo-activated transport and might enable motors to participate in bidirectional cargo transport without undertaking a tug-of-war.


Assuntos
Cinesina/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismo , Proteínas Tirosina Fosfatases não Receptoras/metabolismo , Animais , Transporte Biológico , Linhagem Celular , Vesículas Citoplasmáticas/metabolismo , Hipocampo/citologia , Humanos , Integrinas/metabolismo , Microscopia Intravital/métodos , Cinesina/genética , Cinesina/isolamento & purificação , Camundongos , Proteínas Associadas aos Microtúbulos/isolamento & purificação , Microtúbulos/metabolismo , Neurônios/citologia , Cultura Primária de Células , Ligação Proteica , Domínios Proteicos , Proteínas Tirosina Fosfatases não Receptoras/genética , Proteínas Tirosina Fosfatases não Receptoras/isolamento & purificação , RNA Interferente Pequeno/metabolismo , Ratos , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Imagem Individual de Molécula/métodos
8.
Mol Biol (Mosk) ; 53(3): 446-455, 2019.
Artigo em Russo | MEDLINE | ID: mdl-31184610

RESUMO

Ran is an evolutionarily conserved GTPase crucial in regulating various cell divisions, including mitosis and meiosis. A previous study showed that the knockdown of RAN1 inhibited macronuclear amitosis with the abnormal organization of intramacronuclear microtubules in Tetrahymena thermophila. This study aimed to further investigate the effects of the inducible expression of wild-type Ran1 (Ran1WT), GTP-bound Ran1-mimetic (Ran1Q70L), and GDP-bound Ran1-mimetic (Ran1T25N) on cytoplasmic microtubule assembly during amitosis of T. thermophila, based on previous studies about their effects on intramacronuclear microtubule. The mutant strains of T. thermophila for inducible expression of Ran1WT/T25N/Q70L by Cd^(2+) were constructed. The inducibly expressed HA-Ran1Q70L/T25N distributed asymmetrically across the macronuclear envelope during amitosis. At the lower level of inducible expression, only Ran1T25N showed a significant decreasing effect on T. thermophila reproduction, macronuclear amitosis and cytokinesis. At the higher level of inducible expression, Ran1WT/Q70L/T25N inhibited T. thermophila reproduction, macronuclear amitosis and cytokinesis, and the inhibitive effect of Ran1T25N was the most significant. The inducible expression of Ran1WT/Q70L/T25N led to defects in amitosis and cytokinesis with abnormal cytoplasmic microtubule assembly. These results further confirmed the regulatory function of Ran1 on amitosis and suggested a novel role of Ran1 in cytokinesis and the alignment of cytoplasmic microtubules in T. thermophila.


Assuntos
Citocinese , Guanosina Difosfato/metabolismo , Guanosina Trifosfato/metabolismo , Microtúbulos/metabolismo , Mutação , Proteínas de Protozoários/metabolismo , Tetrahymena thermophila , Proteína ran de Ligação ao GTP/metabolismo , Microtúbulos/patologia , Mitose , Proteínas de Protozoários/genética , Tetrahymena thermophila/citologia , Tetrahymena thermophila/genética , Tetrahymena thermophila/metabolismo , Proteína ran de Ligação ao GTP/genética
9.
Nat Commun ; 10(1): 2208, 2019 05 17.
Artigo em Inglês | MEDLINE | ID: mdl-31101817

RESUMO

Cortical force generators connect epithelial polarity sites with astral microtubules, allowing dynein movement to orient the mitotic spindle as astral microtubules depolymerize. Complexes of the LGN and NuMA proteins, fundamental components of force generators, are recruited to the cortex by Gαi-subunits of heterotrimeric G-proteins. They associate with dynein/dynactin and activate the motor activity pulling on astral microtubules. The architecture of cortical force generators is unknown. Here we report the crystal structure of NuMA:LGN hetero-hexamers, and unveil their role in promoting the assembly of active cortical dynein/dynactin motors that are required in orchestrating oriented divisions in polarized cells. Our work elucidates the basis for the structural organization of essential spindle orientation motors.


Assuntos
Antígenos Nucleares/metabolismo , Polaridade Celular , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas Associadas à Matriz Nuclear/metabolismo , Fuso Acromático/metabolismo , Antígenos Nucleares/química , Antígenos Nucleares/genética , Antígenos Nucleares/isolamento & purificação , Células CACO-2 , Cristalografia por Raios X , Complexo Dinactina/metabolismo , Dineínas/metabolismo , Técnicas de Silenciamento de Genes , Células HEK293 , Células HeLa , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/química , Peptídeos e Proteínas de Sinalização Intracelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/isolamento & purificação , Microtúbulos/metabolismo , Proteínas Associadas à Matriz Nuclear/química , Proteínas Associadas à Matriz Nuclear/genética , Proteínas Associadas à Matriz Nuclear/isolamento & purificação , Ligação Proteica/fisiologia , Multimerização Proteica/fisiologia , RNA Interferente Pequeno/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo
10.
Nat Commun ; 10(1): 2129, 2019 05 13.
Artigo em Inglês | MEDLINE | ID: mdl-31086189

RESUMO

De novo heterozygous missense variants in the γ-tubulin gene TUBG1 have been linked to human malformations of cortical development associated with intellectual disability and epilepsy. Here, we investigated through in-utero electroporation and in-vivo studies, how four of these variants affect cortical development. We show that TUBG1 mutants affect neuronal positioning, disrupting the locomotion of new-born neurons but without affecting progenitors' proliferation. We further demonstrate that pathogenic TUBG1 variants are linked to reduced microtubule dynamics but without major structural nor functional centrosome defects in subject-derived fibroblasts. Additionally, we developed a knock-in Tubg1Y92C/+ mouse model and assessed consequences of the mutation. Although centrosomal positioning in bipolar neurons is correct, they fail to initiate locomotion. Furthermore, Tubg1Y92C/+ animals show neuroanatomical and behavioral defects and increased epileptic cortical activity. We show that Tubg1Y92C/+ mice partially mimic the human phenotype and therefore represent a relevant model for further investigations of the physiopathology of cortical malformations.


Assuntos
Malformações do Desenvolvimento Cortical/genética , Microtúbulos/metabolismo , Neurogênese/genética , Neurônios/fisiologia , Tubulina (Proteína)/genética , Animais , Comportamento Animal , Movimento Celular/genética , Centrossomo/metabolismo , Córtex Cerebral/anormalidades , Córtex Cerebral/citologia , Córtex Cerebral/diagnóstico por imagem , Modelos Animais de Doenças , Embrião de Mamíferos , Epilepsia/genética , Feminino , Fibroblastos/citologia , Fibroblastos/metabolismo , Fibroblastos/ultraestrutura , Técnicas de Introdução de Genes , Predisposição Genética para Doença , Células HeLa , Humanos , Microscopia Intravital , Masculino , Camundongos , Camundongos Transgênicos , Microscopia Confocal , Microscopia Eletrônica , Microtúbulos/genética , Mutação de Sentido Incorreto
11.
J Phys Chem Lett ; 10(11): 3071-3079, 2019 Jun 06.
Artigo em Inglês | MEDLINE | ID: mdl-31117686

RESUMO

Vesicle transport conducted by motor protein multiplexes (MPMs), which is ubiquitous among eukaryotes, shows anomalous and stochastic dynamics qualitatively different from the dynamics of thermal motion and artificial active matter; the relationship between in vivo vesicle-delivery dynamics and the underlying physicochemical processes is not yet quantitatively understood. Addressing this issue, we perform accurate tracking of individual vesicles, containing upconverting nanoparticles, transported by kinesin-dynein-multiplexes along axonal microtubules. The mean-square-displacement of vesicles along the microtubule exhibits unusual dynamic phase transitions that are seemingly inconsistent with the scaling behavior of the mean-first-passage time over the travel length. These paradoxical results and the vesicle displacement distribution are quantitatively explained and predicted by a multimode MPM model, developed in the current work, where ATP-hydrolysis-coupled motion of MPM has both unidirectional and bidirectional modes.


Assuntos
Dineínas/metabolismo , Cinesina/metabolismo , Corpos Multivesiculares/metabolismo , Trifosfato de Adenosina/metabolismo , Transporte Axonal , Transporte Biológico Ativo , Linhagem Celular , Humanos , Hidrólise , Cinética , Microtúbulos/metabolismo , Modelos Biológicos , Nanopartículas/metabolismo
12.
Nat Mater ; 18(6): 638-649, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-31114072

RESUMO

The interrelationship between microtubules and the actin cytoskeleton in mechanoregulation of integrin-mediated adhesions is poorly understood. Here, we show that the effects of microtubules on two major types of cell-matrix adhesion, focal adhesions and podosomes, are mediated by KANK family proteins connecting the adhesion protein talin with microtubule tips. Both total microtubule disruption and microtubule uncoupling from adhesions by manipulations with KANKs trigger a massive assembly of myosin IIA filaments, augmenting focal adhesions and disrupting podosomes. Myosin IIA filaments are indispensable effectors in the microtubule-driven regulation of integrin-mediated adhesions. Myosin IIA filament assembly depends on Rho activation by the RhoGEF GEF-H1, which is trapped by microtubules when they are connected with integrin-mediated adhesions via KANK proteins but released after their disconnection. Thus, microtubule capture by integrin-mediated adhesions modulates the GEF-H1-dependent effect of microtubules on the assembly of myosin IIA filaments. Subsequent actomyosin reorganization then remodels the focal adhesions and podosomes, closing the regulatory loop.


Assuntos
Adesões Focais/metabolismo , Integrinas/metabolismo , Microtúbulos/metabolismo , Miosina não Muscular Tipo IIA/metabolismo , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Linhagem Celular Tumoral , Humanos , Mecanotransdução Celular , Podossomos/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Fatores de Troca de Nucleotídeo Guanina Rho/metabolismo , Proteínas Supressoras de Tumor/genética , Proteínas Supressoras de Tumor/metabolismo , Proteínas rho de Ligação ao GTP/metabolismo , Quinases Associadas a rho/metabolismo
13.
Eur Biophys J ; 48(6): 569-577, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31134309

RESUMO

Microtubule mechanical properties are essential for understanding basic cellular processes, including cell motility and division, but the forces that result in microtubule rupture or breakage have not yet been measured directly. These forces are essential to understand the mechanical properties of the cytoskeleton and responses by cells to both normal conditions and stress caused by injury or disease. Here we estimate the force required to rupture a microtubule by analyzing kinesin-14 Ncd motor-induced microtubule breakage in ensemble motility assays. We model the breakage events as caused by Ncd motors pulling or pushing on single microtubules that are clamped at one end by other motors attached to the glass surface. The number of pulling or pushing Ncd motors is approximated from the length of the microtubule bound to the surface and the forces produced by the pulling or pushing motors are estimated from forces produced by the Ncd motor in laser-trap assays, reported by others. Our analysis provides an estimate, to the first approximation, of ~ 500 pN for the minimal force required to rupture a 13-pf microtubule. The value we report is close to the forces estimated from microtubule stretching/fragmentation experiments and overlaps with the forces applied by AFM in microtubule indentation assays that destabilize microtubules and break microtubule protofilaments. It is also consistent with the forces required to disrupt protein noncovalent bonds in force spectroscopy experiments. These findings are relevant to microtubule deformation and breakage caused by cellular tension in vivo.


Assuntos
Fenômenos Mecânicos , Microtúbulos/metabolismo , Fenômenos Biomecânicos , Ligações de Hidrogênio , Cinesina/metabolismo , Modelos Moleculares , Multimerização Proteica , Estrutura Quaternária de Proteína , Tubulina (Proteína)/química
14.
Nat Commun ; 10(1): 1673, 2019 04 11.
Artigo em Inglês | MEDLINE | ID: mdl-30975984

RESUMO

Accurate chromosome segregation relies on microtubule end conversion, the ill-understood ability of kinetochores to transit from lateral microtubule attachment to durable association with dynamic microtubule plus-ends. The molecular requirements for this conversion and the underlying biophysical mechanisms are elusive. We reconstituted end conversion in vitro using two kinetochore components: the plus end-directed kinesin CENP-E and microtubule-binding Ndc80 complex, combined on the surface of a microbead. The primary role of CENP-E is to ensure close proximity between Ndc80 complexes and the microtubule plus-end, whereas Ndc80 complexes provide lasting microtubule association by diffusing on the microtubule wall near its tip. Together, these proteins mediate robust plus-end coupling during several rounds of microtubule dynamics, in the absence of any specialized tip-binding or regulatory proteins. Using a Brownian dynamics model, we show that end conversion is an emergent property of multimolecular ensembles of microtubule wall-binding proteins with finely tuned force-dependent motility characteristics.


Assuntos
Segregação de Cromossomos , Cinesina/metabolismo , Cinetocoros/metabolismo , Microtúbulos/metabolismo , Animais , Proteínas Cromossômicas não Histona/genética , Proteínas Cromossômicas não Histona/isolamento & purificação , Proteínas Cromossômicas não Histona/metabolismo , Microscopia de Fluorescência , Modelos Biológicos , Dinâmica não Linear , Proteínas Nucleares/genética , Proteínas Nucleares/isolamento & purificação , Proteínas Nucleares/metabolismo , Ligação Proteica , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Células Sf9 , Imagem Individual de Molécula , Processos Estocásticos , Proteínas de Xenopus/genética , Proteínas de Xenopus/isolamento & purificação , Proteínas de Xenopus/metabolismo
15.
Phys Rev E ; 99(3-1): 032411, 2019 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-30999491

RESUMO

Motivated by the recent experimental observations on motor induced cooperative mechanism controlling the length dynamics of microtubules (MTs), we examine how plus-end-targeted proteins of the kinesin family regulate MT polymerization and depolymerization routines. Here, we study a stochastic mathematical model capturing the unusual form of collective motor interaction on MT dynamics originating due to the molecular traffic near the MT tip. We provide an extensive analysis of the joint effect of motor impelled MT polymerization and complete depolymerization. The effect of the cooperative action is included by modifying the intrinsic depolymerization rate. We analyze the model within the framework of continuum mean-field theory and the resultant steady-state analytic solution is expressed in terms of Lambert W functions. Four distinct steady-state phases including a shock phase have been reported. The significant features of the shock including its position and height have been analyzed. Theoretical outcomes are supported by extensive Monte Carlo simulations. To explore the system alterations between the regime of growth and shrinkage phase, we consider kymographs of the microtubule along with the length distributions. Finally, we investigated the dependence of MT length kinetics both on modifying factor of depolymerization rate and motor concentration. The overall extensive study reveals that the flux of molecular traffic at the microtubule plus end initiates a cooperative mechanism, resulting in significant change in MT growth and shrinkage regime as also observed experimentally.


Assuntos
Microtúbulos/metabolismo , Modelos Biológicos , Proteínas Motores Moleculares/metabolismo , Simulação por Computador , Microtúbulos/ultraestrutura , Método de Monte Carlo , Polimerização , Multimerização Proteica , Processos Estocásticos
16.
Parasitol Res ; 118(6): 1899-1918, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-30949853

RESUMO

After host cell invasion, Toxoplasma secretes a variety of dense granule proteins (GRA proteins) from its secretory dense granules, which are involved in the biogenesis of the parasitophorous vacuole (PV). TgGRA8I is predicted to contain proline-rich domains, which are structural features of some cytoskeleton-related proteins. In agreement with this observation, previous proteomic analyses revealed the presence of TgGRA8I in the Toxoplasma sub-pellicular cytoskeleton. In the present study, we show (1) by docking analyses that TgGRA8I may interact with both Toxoplasma ß-tubulin and actin; (2) by immunoelectron microscopy, proteomic, biochemical, and cellular approaches that TgGRA8I associates with sub-pellicular microtubules and actin at the parasite sub-pellicular cytoskeleton; (3) that type I parasites (RH strain) lacking the GRA8 gene (RHΔku80Δgra8) exhibit loss of conoid extrusion, diminished cell infection, and egress capabilities, and that these motility impairments were likely due to important alterations in their sub-pellicular cytoskeleton, in particular their sub-pellicular microtubules and meshwork. Parasites lacking the GRA4 gene (RHΔku80Δgra4) did not show modifications in the organization of the sub-pellicular cytoskeleton. Collectively, these results demonstrated that TgGRA8I is a dense granule protein that, besides its role in the formation of the PV, contributes to the organization of the parasite sub-pellicular cytoskeleton and motility. This is the first proline-rich protein described in the Toxoplasma cytoskeleton, which is a key organelle for both the parasite motility and the invasion process. Knowledge about the function of cytoskeleton components in Toxoplasma is fundamental to understand the motility process and the host cell invasion mechanism. Refining this knowledge should lead to the design of novel pharmacological strategies for the treatment against toxoplasmosis.


Assuntos
Actinas/metabolismo , Antígenos de Protozoários/metabolismo , Movimento Celular/genética , Citoesqueleto/metabolismo , Proteínas de Protozoários/metabolismo , Toxoplasma/metabolismo , Toxoplasma/patogenicidade , Tubulina (Proteína)/metabolismo , Animais , Antígenos de Protozoários/genética , Transporte Biológico , Microscopia Imunoeletrônica , Microtúbulos/metabolismo , Simulação de Acoplamento Molecular , Proteômica , Proteínas de Protozoários/genética , Vesículas Secretórias/metabolismo , Toxoplasma/genética , Toxoplasmose/parasitologia , Toxoplasmose/patologia , Vacúolos/parasitologia
17.
Int J Mol Sci ; 20(8)2019 Apr 20.
Artigo em Inglês | MEDLINE | ID: mdl-31010006

RESUMO

Vincristine is used in the clinical treatment of colon cancer, especially in patients diagnosed in the advanced phase of cancer development. Unfortunately, similar to other agents used during antitumor therapy, vincristine might induce chemoresistance. Studies of this process focus mainly on the analysis of the molecular mechanisms within cancer, usually ignoring the role of stromal cells. Our present findings confirm that vincristine stimulates the secretion of tumor growth factors class beta and interleukin-6 from cancer-associated fibroblasts as a result of paracrine stimulation by cancer cells. Based on alterations in morphology, modulation of capillary formation, and changes in endothelial and mesenchymal marker profile, our findings demonstrate that higher levels of tumor growth factor-ßs and interleukin-6 enhance cancer-associated fibroblast-like cell formation through endothelial-mesenchymal transition and that nonsteroidal anti-inflammatory drug treatment (aspirin and ibuprofen) is able to inhibit this phenomenon. The process appears to be regulated by the rate of microtubule polymerization, depending on ß-tubulin composition. While higher levels of tubulin-ß2 and tubulin-ß4 caused slowed polymerization and reduced the level of factors secreted to the extracellular matrix, tubulin-ß3 induced the opposite effect. We conclude that nonsteroidal anti-inflammatory drugs should be considered for use during vincristine monotherapy in the treatment of patients diagnosed with colorectal cancer.


Assuntos
Anti-Inflamatórios não Esteroides/farmacologia , Fibroblastos Associados a Câncer/patologia , Vincristina/farmacologia , Fibroblastos Associados a Câncer/efeitos dos fármacos , Fibroblastos Associados a Câncer/metabolismo , Comunicação Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Transdiferenciação Celular/efeitos dos fármacos , Neoplasias do Colo/patologia , Meios de Cultivo Condicionados/farmacologia , Citocinas/metabolismo , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/metabolismo , Endotélio/efeitos dos fármacos , Endotélio/patologia , Humanos , Mesoderma/efeitos dos fármacos , Mesoderma/patologia , Microtúbulos/efeitos dos fármacos , Microtúbulos/metabolismo , Polimerização , Tubulina (Proteína)/metabolismo
18.
Biomolecules ; 9(4)2019 04 01.
Artigo em Inglês | MEDLINE | ID: mdl-30939864

RESUMO

The mitotic spindle segregates chromosomes into two daughter cells during cell division. This process relies on the precise regulation of forces acting on chromosomes as the cell progresses through mitosis. The forces in the spindle are difficult to directly measure using the available experimental techniques. Here, we review the ideas and recent advances of how forces can be determined from the spindle shape. By using these approaches, it has been shown that tension and compression coexist along a single kinetochore fiber, which are balanced by a bridging fiber between sister kinetochore fibers. An extension of this approach to three dimensions revealed that microtubule bundles have rich shapes, and extend not simply like meridians on the Earth's surface but, rather, twisted in a helical manner. Such complex shapes are due to rotational forces, which, in addition to linear forces, act in the spindle and may be generated by motor proteins such as kinesin-5. These findings open new questions for future studies, to understand the mechanisms of rotational forces and reveal their biological roles in cells.


Assuntos
Rotação , Fuso Acromático/metabolismo , Animais , Humanos , Cinetocoros/metabolismo , Microtúbulos/metabolismo
19.
Eur J Med Chem ; 173: 1-14, 2019 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-30981112

RESUMO

Further optimization of the trimethoxyphenyl scaffold of parent chalcone compound (2a) by introducing a pyridine ring afforded a series of novel pyridine-chalcone derivatives as potential anti-tubulin agents. All the target compounds were evaluated for their antiproliferative activities. Among them, representative compound 16f exhibited the most potent activity with the IC50 values ranging from 0.023 to 0.045 µM against a panel of cancer cell lines. Further mechanism study results demonstrated that compound 16f effectively inhibited the microtubule polymerization by binding to the colchicine site of tubulin. Moreover, cellular mechanism studies disclosed that 16f caused G2/M phase arrest, induced cell apoptosis and disrupted the intracellular microtubule network. Also, 16f reduced the cell migration and disrupted the capillary-like tube formation of human umbilical vein endothelial cells (HUVECs). Importantly, 16f significantly inhibited tumor growth in H22 xenograft models without apparent toxicity, which was stronger than the reference compound CA-4, indicating that it is worthy to investigate 16f as a potent microtubule-destabilizing agent for cancer therapy.


Assuntos
Antineoplásicos/farmacologia , Chalcona/farmacologia , Desenho de Drogas , Microtúbulos/efeitos dos fármacos , Piridinas/farmacologia , Animais , Antineoplásicos/síntese química , Antineoplásicos/química , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Chalcona/síntese química , Chalcona/química , Relação Dose-Resposta a Droga , Ensaios de Seleção de Medicamentos Antitumorais , Humanos , Células K562 , Masculino , Camundongos , Camundongos Endogâmicos ICR , Microtúbulos/metabolismo , Modelos Moleculares , Estrutura Molecular , Neoplasias Experimentais/tratamento farmacológico , Neoplasias Experimentais/metabolismo , Neoplasias Experimentais/patologia , Piridinas/síntese química , Piridinas/química , Relação Estrutura-Atividade , Cicatrização/efeitos dos fármacos
20.
Int J Mol Sci ; 20(5)2019 Mar 03.
Artigo em Inglês | MEDLINE | ID: mdl-30832428

RESUMO

This study compares the role of electrostatics in the binding process between microtubules and two dynein microtubule-binding domains (MTBDs): cytoplasmic and axonemal. These two dyneins are distinctively different in terms of their functionalities: cytoplasmic dynein is processive, while axonemal dynein is involved in beating. In both cases, the binding requires frequent association/disassociation between the microtubule and MTBD, and involves highly negatively charged microtubules, including non-structured C-terminal domains (E-hooks), and an MTBD interface that is positively charged. This indicates that electrostatics play an important role in the association process. Here, we show that the cytoplasmic MTBD binds electrostatically tighter to microtubules than to the axonemal MTBD, but the axonemal MTBD experiences interactions with microtubule E-hooks at longer distances compared with the cytoplasmic MTBD. This allows the axonemal MTBD to be weakly bound to the microtubule, while at the same time acting onto the microtubule via the flexible E-hooks, even at MTBD⁻microtubule distances of 45 Å. In part, this is due to the charge distribution of MTBDs: in the cytoplasmic MTBD, the positive charges are concentrated at the binding interface with the microtubule, while in the axonemal MTBD, they are more distributed over the entire structure, allowing E-hooks to interact at longer distances. The dissimilarities of electrostatics in the cases of axonemal and cytoplasmic MTBDs were found not to result in a difference in conformational dynamics on MTBDs, while causing differences in the conformational states of E-hooks. The E-hooks' conformations in the presence of the axonemal MTBD were less restricted than in the presence of the cytoplasmic MTBD. In parallel with the differences, the common effect was found that the structural fluctuations of MTBDs decrease as either the number of contacts with E-hooks increases or the distance to the microtubule decreases.


Assuntos
Dineínas do Axonema/química , Dineínas do Citoplasma/química , Simulação de Dinâmica Molecular , Animais , Dineínas do Axonema/metabolismo , Sítios de Ligação , Dineínas do Citoplasma/metabolismo , Camundongos , Microtúbulos/metabolismo , Ligação Proteica
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