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1.
PLoS Biol ; 22(3): e3002551, 2024 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-38466773

RESUMO

Mammals have 6 highly conserved actin isoforms with nonredundant biological functions. The molecular basis of isoform specificity, however, remains elusive due to a lack of tools. Here, we describe the development of IntAct, an internal tagging strategy to study actin isoforms in fixed and living cells. We identified a residue pair in ß-actin that permits tag integration and used knock-in cell lines to demonstrate that IntAct ß-actin expression and filament incorporation is indistinguishable from wild type. Furthermore, IntAct ß-actin remains associated with common actin-binding proteins (ABPs) and can be targeted in living cells. We demonstrate the usability of IntAct for actin isoform investigations by showing that actin isoform-specific distribution is maintained in human cells. Lastly, we observed a variant-dependent incorporation of tagged actin variants into yeast actin patches, cables, and cytokinetic rings demonstrating cross species applicability. Together, our data indicate that IntAct is a versatile tool to study actin isoform localization, dynamics, and molecular interactions.


Assuntos
Actinas , Proteínas dos Microfilamentos , Animais , Humanos , Actinas/genética , Actinas/metabolismo , Proteínas dos Microfilamentos/metabolismo , Citoesqueleto de Actina/metabolismo , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Citoesqueleto/metabolismo , Saccharomyces cerevisiae/metabolismo , Mamíferos/metabolismo
2.
bioRxiv ; 2024 Feb 29.
Artigo em Inglês | MEDLINE | ID: mdl-38464100

RESUMO

Doublecortin (DCX) is a microtubule-associated protein critical for brain development. Although most highly expressed in the developing central nervous system, the molecular function of DCX in neuron morphogenesis remains unknown and controversial. We demonstrate that DCX function is intimately linked to its microtubule-binding activity. By using human induced pluripotent stem cell (hiPSC)- derived cortical i 3 Neurons genome engineered to express mEmerald-tagged DCX from the endogenous locus, we find that DCX-MT interactions become highly polarized very early during neuron morphogenesis. DCX becomes enriched only on straight microtubules in advancing growth cones with approximately 120 DCX molecules bound per micrometer of growth cone microtubule. At a similar saturation, microtubule-bound DCX molecules begin to impede lysosome transport, and thus can potentially control growth cone organelle entry. In addition, by comparing control, DCX-mEmerald and knockout DCX -/Y i 3 Neurons, we find that DCX stabilizes microtubules in the growth cone peripheral domain by reducing the microtubule catastrophe frequency and the depolymerization rate. DCX -/Y i 3 Neuron morphogenesis was inhibited in soft microenvironments that mimic the viscoelasticity of brain tissue and DCX -/Y neurites failed to grow toward brain-derived neurotrophic factor (BDNF) gradients. Together with high resolution traction force microscopy data, we propose a model in which DCX-decorated, rigid growth cone microtubules provide intracellular mechanical resistance to actomyosin generated contractile forces in soft physiological environments in which weak and transient adhesion-mediated forces in the growth cone periphery may be insufficient for productive growth cone advance. These data provide a new mechanistic understanding of how DCX mutations cause lissencephaly-spectrum brain malformations by impacting growth cone dynamics during neuron morphogenesis in physiological environments.

3.
Elife ; 122023 01 30.
Artigo em Inglês | MEDLINE | ID: mdl-36715499

RESUMO

A challenge in analyzing dynamic intracellular cell biological processes is the dearth of methodologies that are sufficiently fast and specific to perturb intracellular protein activities. We previously developed a light-sensitive variant of the microtubule plus end-tracking protein EB1 by inserting a blue light-controlled protein dimerization module between functional domains. Here, we describe an advanced method to replace endogenous EB1 with this light-sensitive variant in a single genome editing step, thereby enabling this approach in human induced pluripotent stem cells (hiPSCs) and hiPSC-derived neurons. We demonstrate that acute and local optogenetic EB1 inactivation in developing cortical neurons induces microtubule depolymerization in the growth cone periphery and subsequent neurite retraction. In addition, advancing growth cones are repelled from areas of blue light exposure. These phenotypes were independent of the neuronal EB1 homolog EB3, revealing a direct dynamic role of EB1-mediated microtubule plus end interactions in neuron morphogenesis and neurite guidance.


Assuntos
Células-Tronco Pluripotentes Induzidas , Proteínas Associadas aos Microtúbulos , Humanos , Genômica , Cones de Crescimento/metabolismo , Células-Tronco Pluripotentes Induzidas/metabolismo , Proteínas Associadas aos Microtúbulos/genética , Proteínas Associadas aos Microtúbulos/metabolismo , Microtúbulos/metabolismo , Ligação Proteica
4.
Methods Mol Biol ; 2430: 467-481, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35476350

RESUMO

Micropatterning of extracellular matrix proteins enables defining cell position and shape in experiments investigating intracellular dynamics and organization. While such standardization is advantageous in automated and quantitative analysis of many cells, the original methods generating such patterns are cumbersome and inflexible. However, recent development of contact-less methods that allow photochemical generation of protein patterns robustly and rapidly is boosting the broader availability of micropatterning approaches. Here, we describe an optimized protocol to achieve large micropatterned areas with high fidelity using a commercially available microscope-mounted UV projection system.


Assuntos
Proteínas da Matriz Extracelular , Matriz Extracelular , Forma Celular , Proteínas da Matriz Extracelular/metabolismo , Microtúbulos/metabolismo
5.
J Cell Biol ; 221(4)2022 04 04.
Artigo em Inglês | MEDLINE | ID: mdl-35266954

RESUMO

Missense mutations in leucine-rich repeat kinase 2 (LRRK2) are the most common cause of familial Parkinson's disease (PD); however, pathways regulating LRRK2 subcellular localization, function, and turnover are not fully defined. We performed quantitative mass spectrometry-based interactome studies to identify 48 novel LRRK2 interactors, including the microtubule-associated E3 ubiquitin ligase TRIM1 (tripartite motif family 1). TRIM1 recruits LRRK2 to the microtubule cytoskeleton for ubiquitination and proteasomal degradation by binding LRRK2911-919, a nine amino acid segment within a flexible interdomain region (LRRK2853-981), which we designate the "regulatory loop" (RL). Phosphorylation of LRRK2 Ser910/Ser935 within LRRK2 RL influences LRRK2's association with cytoplasmic 14-3-3 versus microtubule-bound TRIM1. Association with TRIM1 modulates LRRK2's interaction with Rab29 and prevents upregulation of LRRK2 kinase activity by Rab29 in an E3-ligase-dependent manner. Finally, TRIM1 rescues neurite outgrowth deficits caused by PD-driving mutant LRRK2 G2019S. Our data suggest that TRIM1 is a critical regulator of LRRK2, controlling its degradation, localization, binding partners, kinase activity, and cytotoxicity.


Assuntos
Serina-Treonina Proteína Quinase-2 com Repetições Ricas em Leucina , Doença de Parkinson , Proteínas Serina-Treonina Quinases , Proteínas com Motivo Tripartido , Citoesqueleto , Humanos , Serina-Treonina Proteína Quinase-2 com Repetições Ricas em Leucina/genética , Serina-Treonina Proteína Quinase-2 com Repetições Ricas em Leucina/metabolismo , Proteínas Associadas aos Microtúbulos , Microtúbulos , Mutação , Doença de Parkinson/metabolismo , Fosforilação , Proteínas Serina-Treonina Quinases/genética , Fatores de Transcrição , Proteínas com Motivo Tripartido/metabolismo , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitinação , Proteínas rab de Ligação ao GTP/metabolismo
6.
Curr Biol ; 32(5): 1197-1205.e4, 2022 03 14.
Artigo em Inglês | MEDLINE | ID: mdl-35090591

RESUMO

Chromosome segregation is accomplished by the mitotic spindle, a bipolar micromachine built primarily from microtubules. Different microtubule populations contribute to spindle function: kinetochore microtubules attach and transmit forces to chromosomes, antiparallel interpolar microtubules support spindle structure, and astral microtubules connect spindle poles to the cell cortex.1,2 In mammalian cells, end-binding (EB) proteins associate with all growing microtubule plus ends throughout the cell cycle and serve as adaptors for diverse +TIPs that control microtubule dynamics and interactions with other intracellular structures.3 Because binding of many +TIPs to EB1 and thus microtubule-end association is switched off by mitotic phosphorylation,4-6 the mitotic function of EBs remains poorly understood. To analyze how EB1 and associated +TIPs on different spindle microtubule populations contribute to mitotic spindle dynamics, we use a light-sensitive EB1 variant, π-EB1, that allows local, acute, and reversible inactivation of +TIP association with growing microtubule ends in live cells.7 We find that acute π-EB1 photoinactivation results in rapid and reversible metaphase spindle shortening and transient relaxation of tension across the central spindle. However, in contrast to interphase, π-EB1 photoinactivation does not inhibit microtubule growth in metaphase but instead increases astral microtubule length and number. Yet in the absence of EB1 activity, astral microtubules fail to engage the cortical dynein/dynactin machinery, and spindle poles move away from regions of π-EB1 photoinactivation. In conclusion, our optogenetic approach reveals mitotic EB1 functions that remain hidden in genetic experiments, likely due to compensatory molecular systems regulating vertebrate spindle dynamics.


Assuntos
Proteínas Associadas aos Microtúbulos , Optogenética , Animais , Mamíferos , Metáfase , Proteínas Associadas aos Microtúbulos/genética , Proteínas Associadas aos Microtúbulos/metabolismo , Microtúbulos/metabolismo , Fuso Acromático/metabolismo
7.
J Cell Biol ; 219(9)2020 09 07.
Artigo em Inglês | MEDLINE | ID: mdl-32497170

RESUMO

Microtubule (MT) plus-end tracking proteins (+TIPs) are central players in the coordination between the MT and actin cytoskeletons in growth cones (GCs) during axon guidance. The +TIP Navigator-1 (NAV1) is expressed in the developing nervous system, yet its neuronal functions remain poorly elucidated. Here, we report that NAV1 controls the dynamics and motility of the axonal GCs of cortical neurons in an EB1-dependent manner and is required for axon turning toward a gradient of netrin-1. NAV1 accumulates in F-actin-rich domains of GCs and binds actin filaments in vitro. NAV1 can also bind MTs independently of EB1 in vitro and crosslinks nonpolymerizing MT plus ends to actin filaments in axonal GCs, preventing MT depolymerization in F-actin-rich areas. Together, our findings pinpoint NAV1 as a key player in the actin-MT crosstalk that promotes MT persistence at the GC periphery and regulates GC steering. Additionally, we present data assigning to NAV1 an important role in the radial migration of cortical projection neurons in vivo.


Assuntos
Actinas/metabolismo , Axônios/metabolismo , Cones de Crescimento/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismo , Microtúbulos/metabolismo , Citoesqueleto de Actina/metabolismo , Animais , Orientação de Axônios/fisiologia , Linhagem Celular , Movimento Celular/fisiologia , Feminino , Células HEK293 , Humanos , Camundongos , Netrina-1/metabolismo , Ligação Proteica/fisiologia
8.
Curr Opin Cell Biol ; 66: 1-10, 2020 10.
Artigo em Inglês | MEDLINE | ID: mdl-32371345

RESUMO

Cell biology is moving from observing molecules to controlling them in real time, a critical step towards a mechanistic understanding of how cells work. Initially developed from light-gated ion channels to control neuron activity, optogenetics now describes any genetically encoded protein system designed to accomplish specific light-mediated tasks. Recent photosensitive switches use many ingenious designs that bring spatial and temporal control within reach for almost any protein or pathway of interest. This next generation optogenetics includes light-controlled protein-protein interactions and shape-shifting photosensors, which in combination with live microscopy enable acute modulation and analysis of dynamic protein functions in living cells. We provide a brief overview of various types of optogenetic switches. We then discuss how diverse approaches have been used to control cytoskeleton dynamics with light through Rho GTPase signaling, microtubule and actin assembly, mitotic spindle positioning and intracellular transport and highlight advantages and limitations of different experimental strategies.


Assuntos
Citoesqueleto/metabolismo , Optogenética , Animais , Citoesqueleto/efeitos da radiação , Humanos , Luz , Transdução de Sinais/efeitos da radiação , Proteínas rho de Ligação ao GTP/metabolismo
9.
Methods Mol Biol ; 2101: 211-234, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-31879907

RESUMO

Light can be controlled with high spatial and temporal accuracy. Therefore, optogenetics is an attractive experimental approach to modulate intracellular cytoskeleton dynamics at much faster timescales than by genetic modification. For example, in mammalian cells, microtubules (MTs) grow tens of micrometers per minute and many intracellular MT functions are mediated by a complex of +TIP proteins that dynamically associate with growing MT plus ends. EB1 is a central component of this +TIP protein network, and we recently developed a photo-inactivated π-EB1 by inserting a blue light-sensitive LOV2/Zdk1 module between the EB1 MT-binding domain and the +TIP adaptor domain. Blue light-induced π-EB1 photodissociation results in disassembly of the +TIP complex and strongly attenuates MT growth in mammalian cells.In this chapter, we discuss theoretical and practical aspects of how to perform high-resolution live-cell microscopy in combination with π-EB1 photodissociation. However, these techniques are broadly applicable to other LOV2-based and likely other blue light-sensitive optogenetics. In addition to being a tool to investigate +TIP functions acutely and with subcellular resolution, because of its dramatic and rapid change in intracellular localization, π-EB1 can serve as a powerful tool to test and characterize optogenetic illumination setups. We describe protocols on how to achieve micrometer-scale intracellular control of π-EB1 activity using patterned illumination, and we introduce a do-it-yourself LED cube design compatible with transmitted light microscopy in multiwell plates.


Assuntos
Microscopia de Fluorescência , Microtúbulos/metabolismo , Optogenética , Animais , Linhagem Celular , Humanos , Microscopia de Fluorescência/instrumentação , Microscopia de Fluorescência/métodos , Proteínas Associadas aos Microtúbulos/metabolismo , Optogenética/instrumentação , Optogenética/métodos , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Imagem com Lapso de Tempo
10.
Bioessays ; 41(3): e1800194, 2019 03.
Artigo em Inglês | MEDLINE | ID: mdl-30730055

RESUMO

Microtubules form a highly dynamic filament network in all eukaryotic cells. Individual microtubules grow by tubulin dimer subunit addition and frequently switch between phases of growth and shortening. These unique dynamics are powered by GTP hydrolysis and drive microtubule network remodeling, which is central to eukaryotic cell biology and morphogenesis. Yet, our knowledge of the molecular events at growing microtubule ends remains incomplete. Here, recent ultrastructural, biochemical and cell biological data are integrated to develop a realistic model of growing microtubule ends comprised of structurally distinct but biochemically overlapping zones. Proteins that recognize microtubule lattice conformations associated with specific tubulin guanosine nucleotide states may independently control major structural transitions at growing microtubule ends. A model is proposed in which tubulin dimer addition and subsequent closure of the MT wall are optimized in cells to achieve rapid physiological microtubule growth.


Assuntos
Microtúbulos/metabolismo , Tubulina (Proteína)/química , Animais , Linhagem Celular Tumoral , Microscopia Crioeletrônica , Proteínas do Domínio Duplacortina , Guanosina/química , Guanosina Trifosfato/química , Humanos , Hidrólise , Mamíferos , Proteínas Associadas aos Microtúbulos/metabolismo , Microtúbulos/ultraestrutura , Modelos Moleculares , Morfogênese , Neuropeptídeos/metabolismo , Polimerização , Ligação Proteica , Conformação Proteica , Tubulina (Proteína)/ultraestrutura , Moduladores de Tubulina/metabolismo
11.
Nat Cell Biol ; 20(3): 252-261, 2018 03.
Artigo em Inglês | MEDLINE | ID: mdl-29379139

RESUMO

End-binding proteins (EBs) are adaptors that recruit functionally diverse microtubule plus-end-tracking proteins (+TIPs) to growing microtubule plus ends. To test with high spatial and temporal accuracy how, when and where +TIP complexes contribute to dynamic cell biology, we developed a photo-inactivated EB1 variant (π-EB1) by inserting a blue-light-sensitive protein-protein interaction module between the microtubule-binding and +TIP-binding domains of EB1. π-EB1 replaces endogenous EB1 function in the absence of blue light. By contrast, blue-light-mediated π-EB1 photodissociation results in rapid +TIP complex disassembly, and acutely and reversibly attenuates microtubule growth independent of microtubule end association of the microtubule polymerase CKAP5 (also known as ch-TOG and XMAP215). Local π-EB1 photodissociation allows subcellular control of microtubule dynamics at the second and micrometre scale, and elicits aversive turning of migrating cancer cells. Importantly, light-mediated domain splitting can serve as a template to optically control other intracellular protein activities.


Assuntos
Carcinoma Pulmonar de Células não Pequenas/metabolismo , Movimento Celular , Neoplasias Pulmonares/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismo , Microtúbulos/metabolismo , Optogenética , Carcinoma Pulmonar de Células não Pequenas/genética , Carcinoma Pulmonar de Células não Pequenas/patologia , Linhagem Celular Tumoral , Humanos , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patologia , Microscopia de Fluorescência , Proteínas Associadas aos Microtúbulos/genética , Proteínas Associadas aos Microtúbulos/efeitos da radiação , Microtúbulos/genética , Microtúbulos/patologia , Microtúbulos/efeitos da radiação , Fotólise , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Transdução de Sinais , Fatores de Tempo , Imagem com Lapso de Tempo
12.
J Cell Sci ; 130(8): 1404-1412, 2017 04 15.
Artigo em Inglês | MEDLINE | ID: mdl-28232523

RESUMO

Error-free chromosome segregation requires dynamic control of microtubule attachment to kinetochores, but how kinetochore-microtubule interactions are spatially and temporally controlled during mitosis remains incompletely understood. In addition to the NDC80 microtubule-binding complex, other proteins with demonstrated microtubule-binding activities localize to kinetochores. One such protein is the cytoplasmic linker-associated protein 2 (CLASP2). Here, we show that global GSK3-mediated phosphorylation of the longest isoform, CLASP2α, largely abolishes CLASP2α-microtubule association in metaphase. However, it does not directly control localization of CLASP2α to kinetochores. Using dominant phosphorylation-site variants, we find that CLASP2α phosphorylation weakens kinetochore-microtubule interactions as evidenced by decreased tension between sister kinetochores. Expression of CLASP2α phosphorylation-site mutants also resulted in increased chromosome segregation defects, indicating that GSK3-mediated control of CLASP2α-microtubule interactions contributes to correct chromosome dynamics. Because of global inhibition of CLASP2α-microtubule interactions, we propose a model in which only kinetochore-bound CLASP2α is dephosphorylated, locally engaging its microtubule-binding activity.


Assuntos
Queratinócitos/fisiologia , Cinetocoros/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismo , Microtúbulos/metabolismo , Mitose/fisiologia , Proteína Quinase CDC2 , Linhagem Celular , Segregação de Cromossomos/genética , Quinases Ciclina-Dependentes/metabolismo , Proteínas do Citoesqueleto , Quinase 3 da Glicogênio Sintase/metabolismo , Humanos , Proteínas Associadas aos Microtúbulos/genética , Mutação/genética , Proteínas Nucleares/metabolismo , Fosforilação/genética , Ligação Proteica , Engenharia de Proteínas
13.
Curr Biol ; 26(12): 1549-1555, 2016 06 20.
Artigo em Inglês | MEDLINE | ID: mdl-27238282

RESUMO

Many microtubule (MT) functions are mediated by a diverse class of proteins (+TIPs) at growing MT plus ends that control intracellular MT interactions and dynamics and depend on end-binding proteins (EBs) [1]. Cryoelectron microscopy has recently identified the EB binding site as the interface of four tubulin dimers that undergoes a conformational change in response to ß-tubulin GTP hydrolysis [2, 3]. Doublecortin (DCX), a MT-associated protein (MAP) required for neuronal migration during cortical development [4, 5], binds to the same site as EBs [6], and recent in vitro studies proposed DCX localization to growing MT ends independent of EBs [7]. Because this conflicts with observations in neurons [8, 9] and the molecular function of DCX is not well understood, we revisited intracellular DCX dynamics at low expression levels. Here, we report that DCX is not a +TIP in cells but, on the contrary, is excluded from the EB1 domain. In addition, we find that DCX-MT interactions are highly sensitive to MT geometry. In cells, DCX binding was greatly reduced at MT segments with high local curvature. Remarkably, this geometry-dependent binding to MTs was completely reversed in the presence of taxanes, which reconciles incompatible observations in cells [9] and in vitro [10]. We propose a model explaining DCX specificity for different MT geometries based on structural changes induced by GTP hydrolysis that decreases the spacing between adjacent tubulin dimers [11]. Our data are consistent with a unique mode of MT interaction in which DCX specifically recognizes this compacted GDP-like MT lattice.


Assuntos
Proteínas Associadas aos Microtúbulos/metabolismo , Microtúbulos/metabolismo , Neuropeptídeos/metabolismo , Linhagem Celular Tumoral , Proteínas do Domínio Duplacortina , Proteína Duplacortina , Guanosina Difosfato/metabolismo , Humanos
14.
Curr Biol ; 24(15): 1778-85, 2014 Aug 04.
Artigo em Inglês | MEDLINE | ID: mdl-25065758

RESUMO

Neurite extension is regulated by multiple signaling cascades that ultimately converge on the actin and microtubule networks [1]. Rho GTPases, molecular switches that oscillate between an inactive, GDP-bound state and an active, GTP-bound state, play a pivotal role in controlling actin cytoskeleton dynamics in the growth cone, whereas the dynamic behavior and interactions of microtubules are largely regulated by proteins called plus-end-tracking proteins (+TIPs), which associate with the ends of growing microtubules. Here, we show that the +TIP Navigator 1 (NAV1) is important for neurite outgrowth and interacts and colocalizes with TRIO, a Rho guanine nucleotide exchange factor that enables neurite outgrowth by activating the Rho GTPases Rac1 and RhoG. We find that binding of NAV1 enhances the affinity of TRIO for Rac1 and RhoG, and that NAV1 regulates TRIO-mediated Rac1 activation and neurite outgrowth. TRIO is also a +TIP, as it interacts with the core +TIP EB1 and tracks microtubule plus ends via EB1 and NAV1. Strikingly, the EB1-mediated recruitment of TRIO to microtubule ends is required for proper neurite outgrowth, and stabilization of the microtubule network by paclitaxel affects both the TRIO-NAV1 interaction and the accumulation of these proteins in neurite extensions. We propose that EB1-labeled ends of dynamic microtubules facilitate the formation and localization of functional NAV1-TRIO complexes, which in turn regulate neurite outgrowth by selectively activating Rac1. Our data reveal a novel link between dynamic microtubules, actin cytoskeleton remodeling, and neurite extension.


Assuntos
Proteínas dos Microfilamentos/metabolismo , Fatores de Crescimento Neural/metabolismo , Neuritos/fisiologia , Animais , Linhagem Celular Tumoral , Cones de Crescimento/metabolismo , Células HEK293 , Humanos , Camundongos , Proteínas dos Microfilamentos/genética , Proteínas Associadas aos Microtúbulos , Microtúbulos/metabolismo , Fatores de Crescimento Neural/genética , Ligação Proteica , Transdução de Sinais , Proteínas rac1 de Ligação ao GTP/genética , Proteínas rac1 de Ligação ao GTP/metabolismo , Proteínas rho de Ligação ao GTP/genética , Proteínas rho de Ligação ao GTP/metabolismo
15.
J Cell Sci ; 126(Pt 20): 4589-601, 2013 Oct 15.
Artigo em Inglês | MEDLINE | ID: mdl-23943871

RESUMO

The microtubule (MT) cytoskeleton is essential for many cellular processes, including cell polarity and migration. Cortical platforms, formed by a subset of MT plus-end-tracking proteins, such as CLASP2, and non-MT binding proteins such as LL5ß, attach distal ends of MTs to the cell cortex. However, the mechanisms involved in organizing these platforms have not yet been described in detail. Here we show that 4.1R, a FERM-domain-containing protein, interacts and colocalizes with cortical CLASP2 and is required for the correct number and dynamics of CLASP2 cortical platforms. Protein 4.1R also controls binding of CLASP2 to MTs at the cell edge by locally altering GSK3 activity. Furthermore, in 4.1R-knockdown cells MT plus-ends were maintained for longer in the vicinity of cell edges, but instead of being tethered to the cell cortex, MTs continued to grow, bending at cell margins and losing their radial distribution. Our results suggest a previously unidentified role for the scaffolding protein 4.1R in locally controlling CLASP2 behavior, CLASP2 cortical platform turnover and GSK3 activity, enabling correct MT organization and dynamics essential for cell polarity.


Assuntos
Proteínas do Citoesqueleto/metabolismo , Citoesqueleto/metabolismo , Proteínas de Membrana/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismo , Microtúbulos/metabolismo , Movimento Celular/fisiologia , Polaridade Celular/fisiologia , Técnicas de Silenciamento de Genes , Células HEK293 , Humanos , Proteínas Associadas aos Microtúbulos/genética , Ligação Proteica , Estrutura Terciária de Proteína
16.
Genes Dev ; 27(7): 767-77, 2013 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-23592796

RESUMO

Transcription steps are marked by different modifications of the C-terminal domain of RNA polymerase II (RNAPII). Phosphorylation of Ser5 and Ser7 by cyclin-dependent kinase 7 (CDK7) as part of TFIIH marks initiation, whereas phosphorylation of Ser2 by CDK9 marks elongation. These processes are thought to take place in localized transcription foci in the nucleus, known as "transcription factories," but it has been argued that the observed clusters/foci are mere fixation or labeling artifacts. We show that transcription factories exist in living cells as distinct foci by live-imaging fluorescently labeled CDK9, a kinase known to associate with active RNAPII. These foci were observed in different cell types derived from CDK9-mCherry knock-in mice. We show that these foci are very stable while highly dynamic in exchanging CDK9. Chromatin immunoprecipitation (ChIP) coupled with deep sequencing (ChIP-seq) data show that the genome-wide binding sites of CDK9 and initiating RNAPII overlap on transcribed genes. Immunostaining shows that CDK9-mCherry foci colocalize with RNAPII-Ser5P, much less with RNAPII-Ser2P, and not with CDK12 (a kinase reported to be involved in the Ser2 phosphorylation) or with splicing factor SC35. In conclusion, transcription factories exist in living cells, and initiation and elongation of transcripts takes place in different nuclear compartments.


Assuntos
RNA Polimerase II/metabolismo , Imagem com Lapso de Tempo , Transcrição Gênica , Animais , Células Cultivadas , Quinase 9 Dependente de Ciclina/metabolismo , Células-Tronco Embrionárias/citologia , Células-Tronco Embrionárias/enzimologia , Proteínas Luminescentes/metabolismo , Camundongos , Microscopia de Fluorescência , Estrutura Terciária de Proteína , Transporte Proteico , RNA Polimerase II/química , Proteína Vermelha Fluorescente
17.
Cell Rep ; 2(4): 781-8, 2012 Oct 25.
Artigo em Inglês | MEDLINE | ID: mdl-23084744

RESUMO

Mammalian CLASPs are microtubule plus-end tracking proteins whose essential function as regulators of microtubule behavior has been studied mainly in cultured cells. We show here that absence of murine CLASP2 in vivo results in thrombocytopenia, progressive anemia, and pancytopenia, due to defects in megakaryopoiesis, in erythropoiesis, and in the maintenance of hematopoietic stem cell activity. Furthermore, microtubule stability and organization are affected upon attachment of Clasp2 knockout hematopoietic stem-cell-enriched populations, and these cells do not home efficiently toward their bone marrow niche. Strikingly, CLASP2-deficient hematopoietic stem cells contain severely reduced mRNA levels of c-Mpl, which encodes the thrombopoietin receptor, an essential factor for megakaryopoiesis and hematopoietic stem cell maintenance. Our data suggest that thrombopoietin signaling is impaired in Clasp2 knockout mice. We propose that the CLASP2-mediated stabilization of microtubules is required for proper attachment, homing, and maintenance of hematopoietic stem cells and that this is necessary to sustain c-Mpl transcription.


Assuntos
Hematopoese/fisiologia , Células-Tronco Hematopoéticas/citologia , Proteínas Associadas aos Microtúbulos/metabolismo , Microtúbulos/metabolismo , Animais , Células-Tronco Hematopoéticas/metabolismo , Camundongos , Camundongos Knockout , Proteínas Associadas aos Microtúbulos/deficiência , Proteínas Associadas aos Microtúbulos/genética , Transdução de Sinais , Trombopoetina/genética , Trombopoetina/metabolismo
18.
J Cell Biol ; 198(3): 421-37, 2012 Aug 06.
Artigo em Inglês | MEDLINE | ID: mdl-22851317

RESUMO

Agrin is the major factor mediating the neuronal regulation of postsynaptic structures at the vertebrate neuromuscular junction, but the details of how it orchestrates this unique three-dimensional structure remain unknown. Here, we show that agrin induces the formation of the dense network of microtubules in the subsynaptic cytoplasm and that this, in turn, regulates acetylcholine receptor insertion into the postsynaptic membrane. Agrin acted in part by locally activating phosphatidylinositol 3-kinase and inactivating GSK3ß, which led to the local capturing of dynamic microtubules at agrin-induced acetylcholine receptor (AChR) clusters, mediated to a large extent by the microtubule plus-end tracking proteins CLASP2 and CLIP-170. Indeed, in the absence of CLASP2, microtubule plus ends at the subsynaptic muscle membrane, the density of synaptic AChRs, the size of AChR clusters, and the numbers of subsynaptic muscle nuclei with their selective gene expression programs were all reduced. Thus, the cascade linking agrin to CLASP2-mediated microtubule capturing at the synaptic membrane is essential for the maintenance of a normal neuromuscular phenotype.


Assuntos
Agrina/fisiologia , Regulação da Expressão Gênica , Proteínas Associadas aos Microtúbulos/metabolismo , Microtúbulos/metabolismo , Junção Neuromuscular/metabolismo , Membranas Sinápticas/metabolismo , Agrina/química , Animais , Células COS , Linhagem Celular , Chlorocebus aethiops , Deleção de Genes , Quinase 3 da Glicogênio Sintase/metabolismo , Glicogênio Sintase Quinase 3 beta , Proteínas de Fluorescência Verde/metabolismo , Camundongos , Camundongos Knockout , Proteínas Associadas aos Microtúbulos/genética , Modelos Biológicos , Proteínas de Neoplasias/metabolismo , Fenótipo , Fosfatidilinositol 3-Quinases/metabolismo , Fosforilação , Receptores Colinérgicos/metabolismo
19.
Mol Biol Cell ; 21(15): 2661-73, 2010 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-20519438

RESUMO

Cytoplasmic linker protein (CLIP)-170 is a microtubule (MT) plus-end-tracking protein that regulates MT dynamics and links MT plus ends to different intracellular structures. We have shown previously that intramolecular association between the N and C termini results in autoinhibition of CLIP-170, thus altering its binding to MTs and the dynactin subunit p150(Glued) (J. Cell Biol. 2004: 166, 1003-1014). In this study, we demonstrate that conformational changes in CLIP-170 are regulated by phosphorylation that enhances the affinity between the N- and C-terminal domains. By using site-directed mutagenesis and phosphoproteomic analysis, we mapped the phosphorylation sites in the third serine-rich region of CLIP-170. A phosphorylation-deficient mutant of CLIP-170 displays an "open" conformation and a higher binding affinity for growing MT ends and p150(Glued) as compared with nonmutated protein, whereas a phosphomimetic mutant confined to the "folded back" conformation shows decreased MT association and does not interact with p150(Glued). We conclude that phosphorylation regulates CLIP-170 conformational changes resulting in its autoinhibition.


Assuntos
Proteínas Associadas aos Microtúbulos/antagonistas & inibidores , Proteínas de Neoplasias/antagonistas & inibidores , Sequência de Aminoácidos , Animais , Linhagem Celular , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Dineínas/metabolismo , Camundongos , Proteínas Associadas aos Microtúbulos/química , Proteínas Associadas aos Microtúbulos/metabolismo , Microtúbulos/efeitos dos fármacos , Microtúbulos/metabolismo , Modelos Biológicos , Dados de Sequência Molecular , Mutação/genética , Proteínas de Neoplasias/química , Proteínas de Neoplasias/metabolismo , Ácido Okadáico/farmacologia , Fosforilação/efeitos dos fármacos , Fosfosserina/metabolismo , Dobramento de Proteína/efeitos dos fármacos , Transporte Proteico/efeitos dos fármacos , Proteoma/metabolismo
20.
Curr Biol ; 20(11): 1023-8, 2010 Jun 08.
Artigo em Inglês | MEDLINE | ID: mdl-20471267

RESUMO

In Chinese hamster ovary cells, microtubules originate at the microtubule organizing center (MTOC) and grow persistently toward the cell edge, where they undergo catastrophe. In axons, microtubule dynamics must be regulated differently because microtubules grow parallel to the plasma membrane and there is no MTOC. GFP-tagged microtubule plus end tracking proteins (+TIPs) mark the ends of growing neuronal microtubules. Their fluorescent "comet-like" pattern reflects turnover of +TIP binding sites. Using GFP-tagged +TIPs and fluorescence-based segmentation and tracking tools, we show that axonal microtubules grow with a constant average velocity and that they undergo catastrophes at random positions, yet in a programmed fashion. Using protein depletion approaches, we find that the +TIPs CLIP-115 and CLIP-170 affect average microtubule growth rate and growth distance in neurons but not the duration of a microtubule growth event. In N1E-115 neuroblastoma cells, we find that EB1, the core +TIP, regulates microtubule growth rate, growth distance, and duration, consistent with in vitro data. Combined, our data suggest that CLIPs influence the axonal microtubule/tubulin ratio, whereas EB1 stimulates microtubule growth and structural transitions at microtubule ends, thereby regulating microtubule catastrophes and the turnover of +TIP binding sites.


Assuntos
Axônios/ultraestrutura , Microtúbulos/metabolismo , Animais , Axônios/metabolismo , Sítios de Ligação , Células CHO , Linhagem Celular , Cricetinae , Cricetulus , Técnicas de Silenciamento de Genes , Proteínas de Fluorescência Verde/metabolismo , Camundongos , Proteínas Associadas aos Microtúbulos/genética , Proteínas Associadas aos Microtúbulos/metabolismo , Neurônios/citologia , Ligação Proteica , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo
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