RESUMO
Fibroblast to myofibroblast transdifferentiation mediates numerous fibrotic disorders, such as idiopathic pulmonary fibrosis (IPF). We have previously demonstrated that non-muscle myosin II (NMII) is activated in response to fibrotic lung extracellular matrix, thereby mediating myofibroblast transdifferentiation. NMII-A is known to interact with the calcium-binding protein S100A4, but the mechanism by which S100A4 regulates fibrotic disorders is unclear. In this study, we show that fibroblast S100A4 is a calcium-dependent, mechanoeffector protein that is uniquely sensitive to pathophysiologic-range lung stiffness (8-25 kPa) and thereby mediates myofibroblast transdifferentiation. Re-expression of endogenous fibroblast S100A4 rescues the myofibroblastic phenotype in S100A4 KO fibroblasts. Analysis of NMII-A/actin dynamics reveals that S100A4 mediates the unraveling and redistribution of peripheral actomyosin to a central location, resulting in a contractile myofibroblast. Furthermore, S100A4 loss protects against murine in vivo pulmonary fibrosis, and S100A4 expression is dysregulated in IPF. Our data reveal a novel mechanosensor/effector role for endogenous fibroblast S100A4 in inducing cytoskeletal redistribution in fibrotic disorders such as IPF.
Assuntos
Fibrose Pulmonar Idiopática , Mecanotransdução Celular , Miofibroblastos , Proteína A4 de Ligação a Cálcio da Família S100 , Animais , Camundongos , Transdiferenciação Celular , Fibrose , Fibrose Pulmonar Idiopática/metabolismo , Fibrose Pulmonar Idiopática/patologia , Pulmão/metabolismo , Miofibroblastos/metabolismo , Miofibroblastos/patologia , Proteína A4 de Ligação a Cálcio da Família S100/genética , Proteína A4 de Ligação a Cálcio da Família S100/metabolismoRESUMO
Two modes of motility have been reported for bi-directional kinesin-5 motors: (a) context-dependent directionality reversal, a mode in which motors undergo persistent minus-end directed motility at the single-molecule level and switch to plus-end directed motility in different assays or under different conditions, such as during MT gliding or antiparallel sliding or as a function of motor clustering; and (b) bi-directional motility, defined as movement in two directions in the same assay, without persistent unidirectional motility. Here, we examine how modulation of motor-microtubule (MT) interactions affects these two modes of motility for the bi-directional kinesin-5, Cin8. We report that the large insert in loop 8 (L8) within the motor domain of Cin8 increases the MT affinity of Cin8 in vivo and in vitro and is required for Cin8 intracellular functions. We consistently found that recombinant purified L8 directly binds MTs and L8 induces single Cin8 motors to behave according to context-dependent directionality reversal and bi-directional motility modes at intermediate ionic strength and according to a bi-directional motility mode in an MT surface-gliding assay under low motor density conditions. We propose that the largely unstructured L8 facilitates flexible anchoring of Cin8 to the MTs. This flexible anchoring enables the direct observation of bi-directional motility in motility assays. Remarkably, although L8-deleted Cin8 variants exhibit a strong minus-end directed bias at the single-molecule level, they also exhibit plus-end directed motility in an MT-gliding assay. Thus, L8-induced flexible MT anchoring is required for bi-directional motility of single Cin8 molecules but is not necessary for context-dependent directionality reversal of Cin8 in an MT-gliding assay.
Assuntos
Cinesinas/metabolismo , Microtúbulos/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Sequência de Aminoácidos , Movimento/fisiologia , Saccharomyces cerevisiae/metabolismoRESUMO
The ability of glioblastoma to disperse through the brain contributes to its lethality, and blocking this behavior has been an appealing therapeutic approach. Although a number of proinvasive signaling pathways are active in glioblastoma, many are redundant, so targeting one can be overcome by activating another. However, these pathways converge on nonredundant components of the cytoskeleton, and we have shown that inhibiting one of these-the myosin II family of cytoskeletal motors-blocks glioblastoma invasion even with simultaneous activation of multiple upstream promigratory pathways. Myosin IIA and IIB are the most prevalent isoforms of myosin II in glioblastoma, and we now show that codeleting these myosins markedly impairs tumorigenesis and significantly prolongs survival in a rodent model of this disease. However, while targeting just myosin IIA also impairs tumor invasion, it surprisingly increases tumor proliferation in a manner that depends on environmental mechanics. On soft surfaces myosin IIA deletion enhances ERK1/2 activity, while on stiff surfaces it enhances the activity of NFκB, not only in glioblastoma but in triple-negative breast carcinoma and normal keratinocytes as well. We conclude myosin IIA suppresses tumorigenesis in at least two ways that are modulated by the mechanics of the tumor and its stroma. Our results also suggest that inhibiting tumor invasion can enhance tumor proliferation and that effective therapy requires targeting cellular components that drive both proliferation and invasion simultaneously.
Assuntos
Carcinogênese/metabolismo , Citoesqueleto/metabolismo , Glioblastoma/metabolismo , Sistema de Sinalização das MAP Quinases , Proteínas de Neoplasias/metabolismo , Miosina não Muscular Tipo IIA/metabolismo , Animais , Carcinogênese/genética , Carcinogênese/patologia , Linhagem Celular Tumoral , Citoesqueleto/genética , Citoesqueleto/patologia , Glioblastoma/genética , Glioblastoma/patologia , Camundongos , Proteínas de Neoplasias/genética , Miosina não Muscular Tipo IIA/genéticaRESUMO
Numerous posttranslational modifications have been described in kinesins, but their consequences on motor mechanics are largely unknown. We investigated one of these-acetylation of lysine 146 in Eg5-by creating an acetylation mimetic lysine to glutamine substitution (K146Q). Lysine 146 is located in the α2 helix of the motor domain, where it makes an ionic bond with aspartate 91 on the neighboring α1 helix. Molecular dynamics simulations predict that disrupting this bond enhances catalytic site-neck linker coupling. We tested this using structural kinetics and single-molecule mechanics and found that the K146Q mutation increases motor performance under load and coupling of the neck linker to catalytic site. These changes convert Eg5 from a motor that dissociates from the microtubule at low load into one that is more tightly coupled and dissociation resistant-features shared by kinesin 1. These features combined with the increased propensity to stall predict that the K146Q Eg5 acetylation mimetic should act in the cell as a "brake" that slows spindle pole separation, and we have confirmed this by expressing this modified motor in mitotically active cells. Thus, our results illustrate how a posttranslational modification of a kinesin can be used to fine tune motor behavior to meet specific physiological needs.
Assuntos
Cinesinas/química , Cinesinas/metabolismo , Mitose/fisiologia , Sequência de Aminoácidos , Fenômenos Biomecânicos , Células HeLa , Humanos , Modelos Moleculares , Mutação , Conformação ProteicaRESUMO
Kinesins perform mechanical work to power a variety of cellular functions, from mitosis to organelle transport. Distinct functions shape distinct enzymologies, and this is illustrated by comparing kinesin-1, a highly processive transport motor that can work alone, to Eg5, a minimally processive mitotic motor that works in large ensembles. Although crystallographic models for both motors reveal similar structures for the domains involved in mechanochemical transduction--including switch-1 and the neck linker--how movement of these two domains is coordinated through the ATPase cycle remains unknown. We have addressed this issue by using a novel combination of transient kinetics and time-resolved fluorescence, which we refer to as "structural kinetics," to map the timing of structural changes in the switch-1 loop and neck linker. We find that differences between the structural kinetics of Eg5 and kinesin-1 yield insights into how these two motors adapt their enzymologies for their distinct functions.
Assuntos
Cinesinas/fisiologia , Modelos Moleculares , Adenosina Trifosfatases/química , Trifosfato de Adenosina/química , Animais , Sítios de Ligação , Cristalografia por Raios X , Transferência Ressonante de Energia de Fluorescência , Humanos , Cinesinas/química , Cinética , Microtúbulos/química , Ligação Proteica , Multimerização Proteica , Estrutura Terciária de Proteína , Ovinos , TemperaturaRESUMO
Pro-fibrotic mesenchymal cells are known to be the key effector cells of fibroproliferative disease, but the specific matrix signals and the induced cellular responses that drive the fibrogenic phenotype remain to be elucidated. The key mediators of the fibroblast fibrogenic phenotype were characterized using a novel assay system that measures fibroblast behavior in response to actual normal and fibrotic lung tissue. Using this system, we demonstrate that normal lung promotes fibroblast motility and polarization, while fibrotic lung immobilizes the fibroblast and promotes myofibroblast differentiation. These context-specific phenotypes are surprisingly both mediated by myosin II. The role of myosin II is supported by the observation of an increase in myosin phosphorylation and a change in intracellular distribution in fibroblasts on fibrotic lung, as compared with normal lung. Moreover, loss of myosin II activity has opposing effects on protrusive activity in fibroblasts on normal and fibrotic lung. Loss of myosin II also selectively inhibits myofibroblast differentiation in fibroblasts on fibrotic lung. Importantly, these findings are recapitulated by varying the matrix stiffness of polyacrylamide gels in the range of normal and fibrotic lung tissue. Comparison of the effects of myosin inhibition on lung tissue with that of polyacrylamide gels suggests that matrix fiber organization drives the fibroblast phenotype under conditions of normal/soft lung, while matrix stiffness drives the phenotype under conditions of fibrotic/stiff lung. This work defines novel roles for myosin II as a key regulatory effector molecule of the pro-fibrotic phenotype, in response to biophysical properties of the matrix.
Assuntos
Fibroblastos/fisiologia , Miosina Tipo II/fisiologia , Fibrose Pulmonar/metabolismo , Animais , Diferenciação Celular , Linhagem Celular , Movimento Celular , Polaridade Celular , Forma Celular , Matriz Extracelular/fisiologia , Feminino , Humanos , Pulmão/metabolismo , Pulmão/patologia , Camundongos Endogâmicos C57BL , Fenótipo , Fibrose Pulmonar/patologiaRESUMO
Kinesins are responsible for a wide variety of microtubule-based, ATP-dependent functions. Their motor domain drives these activities, but the molecular adaptations that specify these diverse and essential cellular activities are poorly understood. It has been assumed that the first identified kinesin--the transport motor kinesin-1--is the mechanistic paradigm for the entire superfamily, but accumulating evidence suggests otherwise. To address the deficits in our understanding of the molecular basis of functional divergence within the kinesin superfamily, we studied kinesin-5s, which are essential mitotic motors whose inhibition blocks cell division. Using cryo-electron microscopy and determination of structure at subnanometer resolution, we have visualized conformations of microtubule-bound human kinesin-5 motor domain at successive steps in its ATPase cycle. After ATP hydrolysis, nucleotide-dependent conformational changes in the active site are allosterically propagated into rotations of the motor domain and uncurling of the drug-binding loop L5. In addition, the mechanical neck-linker element that is crucial for motor stepping undergoes discrete, ordered displacements. We also observed large reorientations of the motor N terminus that indicate its importance for kinesin-5 function through control of neck-linker conformation. A kinesin-5 mutant lacking this N terminus is enzymatically active, and ATP-dependent neck-linker movement and motility are defective, although not ablated. All these aspects of kinesin-5 mechanochemistry are distinct from kinesin-1. Our findings directly demonstrate the regulatory role of the kinesin-5 N terminus in collaboration with the motor's structured neck-linker and highlight the multiple adaptations within kinesin motor domains that tune their mechanochemistries according to distinct functional requirements.
Assuntos
Cinesinas/química , Cinesinas/metabolismo , Mitose , Modelos Moleculares , Difosfato de Adenosina/metabolismo , Trifosfato de Adenosina/metabolismo , Humanos , Hidrólise , Cinética , Microtúbulos/metabolismo , Ligação Proteica , Estrutura Terciária de Proteína , Deleção de Sequência , Relação Estrutura-AtividadeRESUMO
BACKGROUND: Treatment options for patients with non-small cell lung cancer (NSCLC) with brain metastases are limited. Patupilone (EPO906), a blood-brain barrier-penetrating, microtubule-targeting, cytotoxic agent, has shown clinical activity in phase 1/2 studies in patients with NSCLC. This study evaluates the efficacy, pharmacokinetics, and safety of patupilone in NSCLC brain metastases. METHODS: Adult patients with NSCLC and confirmed progressive brain metastases received patupilone intravenously at 10 mg/m(2) every 3 weeks. The primary endpoint of this multinomial 2-stage study combined early progression (EP; death or progression within 3 weeks) and progression-free survival at 9 weeks (PFS9w) to determine drug activity. RESULTS: Fifty patients with a median age of 60 years (range, 33-74 years) were enrolled; the majority were men (58%), and most had received prior therapy for brain metastases (98%). The PFS9w rate was 36%, and the EP rate was 26%. Patupilone blood pharmacokinetic analyses showed mean areas under the concentration-time curve from time zero to 504 hours for cycles 1 and 3 of 1544 and 1978 ng h/mL, respectively, and a mean steady state distribution volume of 755 L/m(2) . Grade 3/4 adverse events (AEs), regardless of their relation with the study drug, included diarrhea (24%), pulmonary embolisms (8%), convulsions (4%), and peripheral neuropathy (4%). All patients discontinued the study drug: 31 (62%) for disease progression and 13 (26%) for AEs. Twenty-five of 32 deaths were due to brain metastases. The median time to progression and the overall survival were 3.2 and 8.8 months, respectively. CONCLUSIONS: This is the first prospective study of chemotherapy for recurrent brain metastases from NSCLC. In this population, patupilone demonstrated activity in heavily treated patients.
Assuntos
Antineoplásicos/administração & dosagem , Neoplasias Encefálicas/tratamento farmacológico , Neoplasias Encefálicas/secundário , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Epotilonas/administração & dosagem , Neoplasias Pulmonares/tratamento farmacológico , Recidiva Local de Neoplasia/tratamento farmacológico , Administração Intravenosa , Adulto , Idoso , Antineoplásicos/efeitos adversos , Neoplasias Encefálicas/mortalidade , Carcinoma Pulmonar de Células não Pequenas/mortalidade , Progressão da Doença , Esquema de Medicação , Epotilonas/efeitos adversos , Feminino , Humanos , Neoplasias Pulmonares/mortalidade , Masculino , Pessoa de Meia-Idade , Recidiva Local de Neoplasia/mortalidade , Estudos Prospectivos , Análise de Sobrevida , Resultado do TratamentoRESUMO
BACKGROUND: Time trends in cancer incidence rates (IR) are important to measure the changing burden of cancer on a population over time. The overall IR of cancer in the United States is declining. Although central nervous system tumors (CNST) are rare, they contribute disproportionately to mortality and morbidity. In this analysis, the authors examined trends in the incidence of the most common cancers and CNST between 2000 and 2010. METHODS: The current analysis used data from the United States Cancer Statistics publication and the Central Brain Tumor Registry of the United States. Age-adjusted IR per 100,000 population with 95% confidence intervals and the annual percent change (APC) with 95% confidence intervals were calculated for selected common cancers and CNST overall and by age, sex, race/ethnicity, selected histologies, and malignancy status. RESULTS: In adults, there were significant decreases in colon (2000-2010: APC, -3.1), breast (2000-2010: APC, -0.8), lung (2000-2010: APC, -1.1), and prostate (2000-2010: APC, -2.4) cancer as well as malignant CNST (2008-2010: APC, -3.1), but a significant increase was noted in nonmalignant CNST (2004-2010: APC, 2.7). In adolescents, there were significant increases in malignant CNST (2000-2008: APC, 1.0) and nonmalignant CNST (2004-2010: APC, 3.9). In children, there were significant increases in acute lymphocytic leukemia (2000-2010: APC, 1.0), non-Hodgkin lymphoma (2000-2010: APC, 0.6), and malignant CNST (2000-2010: APC, 0.6). CONCLUSIONS: Surveillance of IR trends is an important way to measure the changing public health and economic burden of cancer. In the current study, there were significant decreases noted in the incidence of adult cancer, whereas adolescent and childhood cancer IR were either stable or increasing.
Assuntos
Neoplasias do Sistema Nervoso Central/epidemiologia , Neoplasias/epidemiologia , Adolescente , Adulto , Idoso , Neoplasias do Sistema Nervoso Central/etnologia , Criança , Pré-Escolar , Análise por Conglomerados , Estudos de Coortes , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Neoplasias/etnologia , Programa de SEER , Fatores de Tempo , Estados Unidos/epidemiologia , Estados Unidos/etnologia , Adulto JovemRESUMO
Kinesins comprise a superfamily of molecular motors that drive a wide variety of cellular physiologies, from cytoplasmic transport to formation of the bipolar spindle in mitosis. These differing roles are reflected in corresponding polymorphisms in key kinesin structural elements. One of these is a unique loop and stem motif found in all kinesins and referred to as loop 5 (L5). This loop is longest in the mitotic kinesin Eg5 and is the target for a number of small molecule inhibitors, including ispinesib, which is being used in clinical trials in patients with cancer. In this study, we have used x-ray crystallography to identify a new structure of an Eg5-ispinesib complex and have combined this with transient state kinetics to identify a plausible sequence of conformational changes that occur in response to ispinesib binding. Our results demonstrate that ispinesib-induced structural changes in L5 from Eg5 lead to subsequent changes in the conformation of the switch II loop and helix and in the neck linker. We conclude that L5 in Eg5 simultaneously regulates the structure of both the ATP binding site and the motor's mechanical elements that generate force.
Assuntos
Benzamidas/química , Cinesinas/química , Estrutura Terciária de Proteína , Quinazolinas/química , Algoritmos , Benzamidas/metabolismo , Benzamidas/farmacologia , Sítios de Ligação , Biocatálise/efeitos dos fármacos , Cristalografia por Raios X , Humanos , Cinesinas/genética , Cinesinas/metabolismo , Cinética , Mitose , Modelos Moleculares , Estrutura Molecular , Ligação Proteica , Conformação Proteica/efeitos dos fármacos , Estrutura Secundária de Proteína , Quinazolinas/metabolismo , Quinazolinas/farmacologiaRESUMO
Members of the kinesin superfamily of molecular motors differ in several key structural domains, which probably allows these molecular motors to serve the different physiologies required of them. One of the most variable of these is a stem-loop motif referred to as L5. This loop is longest in the mitotic kinesin Eg5, and previous structural studies have shown that it can assume different conformations in different nucleotide states. However, enzymatic domains often consist of a mixture of conformations whose distribution shifts in response to substrate binding or product release, and this information is not available from the "static" images that structural studies provide. We have addressed this issue in the case of Eg5 by attaching a fluorescent probe to L5 and examining its fluorescence, using both steady state and time-resolved methods. This reveals that L5 assumes an equilibrium mixture of three orientations that differ in their local environment and segmental mobility. Combining these studies with transient state kinetics demonstrates that there is a major shift in this distribution during transitions that interconvert weak and strong microtubule binding states. Finally, in conjunction with previous cryo-EM reconstructions of Eg5·microtubule complexes, these fluorescence studies suggest a model in which L5 regulates both nucleotide and microtubule binding through a set of reversible interactions with helix α3. We propose that these features facilitate the production of sustained opposing force by Eg5, which underlies its role in supporting formation of a bipolar spindle in mitosis.
Assuntos
Adenosina Trifosfatases/metabolismo , Cinesinas/química , Microtúbulos/ultraestrutura , Mitose/genética , Adenosina Trifosfatases/química , Adenilil Imidodifosfato/química , Sítios de Ligação , Humanos , Interações Hidrofóbicas e Hidrofílicas , Cinesinas/genética , Cinesinas/ultraestrutura , Cinética , Microscopia Eletrônica , Microtúbulos/química , Ligação Proteica/genética , Estrutura Secundária de Proteína , Estrutura Terciária de ProteínaRESUMO
Glioblastoma (GBM) is a deadly brain tumor, and the kinesin motor KIF11 is an attractive therapeutic target with roles in proliferation and invasion. Resistance to KIF11 inhibitors, which has mainly been studied in animal models, presents significant challenges. We use lineage-tracing barcodes and single-cell RNA sequencing to analyze resistance in patient-derived GBM neurospheres treated with ispinesib, a potent KIF11 inhibitor. Similar to GBM progression in patients, untreated cells lose their neural lineage identity and become mesenchymal, which is associated with poor prognosis. Conversely, cells subjected to long-term ispinesib treatment exhibit a proneural phenotype. We generate patient-derived xenografts and show that ispinesib-resistant cells form less aggressive tumors in vivo, even in the absence of drug. Moreover, treatment of human ex vivo GBM slices with ispinesib demonstrates phenotypic alignment with in vitro responses, underscoring the clinical relevance of our findings. Finally, using retrospective lineage tracing, we identify drugs that are synergistic with ispinesib.
Assuntos
Linhagem da Célula , Resistencia a Medicamentos Antineoplásicos , Glioblastoma , Cinesinas , Análise de Célula Única , Humanos , Glioblastoma/patologia , Glioblastoma/genética , Glioblastoma/metabolismo , Glioblastoma/tratamento farmacológico , Cinesinas/metabolismo , Cinesinas/antagonistas & inibidores , Cinesinas/genética , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos/genética , Animais , Linhagem da Célula/efeitos dos fármacos , Camundongos , Neoplasias Encefálicas/patologia , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/tratamento farmacológico , Neoplasias Encefálicas/metabolismo , Linhagem Celular Tumoral , Mitose/efeitos dos fármacosRESUMO
While mitotic spindle inhibitors specifically kill proliferating tumor cells without the toxicities of microtubule poisons, resistance has limited their clinical utility. Treating glioblastomas with the spindle inhibitors ispinesib, alisertib, or volasertib creates a subpopulation of therapy induced senescent cells that resist these drugs by relying upon the anti-apoptotic and metabolic effects of activated STAT3. Furthermore, these senescent cells expand the repertoire of cells resistant to these drugs by secreting an array of factors, including TGFß, which induce proliferating cells to exit mitosis and become quiescent-a state that also resists spindle inhibitors. Targeting STAT3 restores sensitivity to each of these drugs by depleting the senescent subpopulation and inducing quiescent cells to enter the mitotic cycle. These results support a therapeutic strategy of targeting STAT3-dependent therapy-induced senescence to enhance the efficacy of spindle inhibitors for the treatment of glioblastoma. Highlights: ⢠Resistance to non-microtubule spindle inhibitors limits their efficacy in glioblastoma and depends on STAT3.⢠Resistance goes hand in hand with development of therapy induced senescence (TIS).⢠Spindle inhibitor resistant glioblastomas consist of three cell subpopulations-proliferative, quiescent, and TIS-with proliferative cells sensitive and quiescent and TIS cells resistant.⢠TIS cells secrete TGFß, which induces proliferative cells to become quiescent, thereby expanding the population of resistant cells in a spindle inhibitor resistant glioblastoma⢠Treatment with a STAT3 inhibitor kills TIS cells and restores sensitivity to spindle inhibitors.
RESUMO
We have identified a NMIIA and IIB-specific small molecule inhibitor, MT-125, and have studied its effects in GBM. MT-125 has high brain penetrance and retention and an excellent safety profile; blocks GBM invasion and cytokinesis, consistent with the known roles of NMII; and prolongs survival as a single agent in murine GBM models. MT-125 increases signaling along both the PDGFR- and MAPK-driven pathways through a mechanism that involves the upregulation of reactive oxygen species, and it synergizes with FDA-approved PDGFR and mTOR inhibitors in vitro . Combining MT-125 with sunitinib, a PDGFR inhibitor, or paxalisib, a combined PI3 Kinase/mTOR inhibitor significantly improves survival in orthotopic GBM models over either drug alone, and in the case of sunitinib, markedly prolongs survival in â¼40% of mice. Our results provide a powerful rationale for developing NMII targeting strategies to treat cancer and demonstrate that MT-125 has strong clinical potential for the treatment of GBM. Highlights: MT-125 is a highly specific small molecule inhibitor of non-muscle myosin IIA and IIB, is well-tolerated, and achieves therapeutic concentrations in the brain with systemic dosing.Treating preclinical models of glioblastoma with MT-125 produces durable improvements in survival.MT-125 stimulates PDGFR- and MAPK-driven signaling in glioblastoma and increases dependency on these pathways.Combining MT-125 with an FDA-approved PDGFR inhibitor in a mouse GBM model synergizes to improve median survival over either drug alone, and produces tumor free, prolonged survival in over 40% of mice.
RESUMO
Gliomas are highly aggressive brain tumors characterized by poor prognosis and composed of diffusely infiltrating tumor cells that intermingle with non-neoplastic cells in the tumor microenvironment, including neurons. Neurons are increasingly appreciated as important reactive components of the glioma microenvironment, due to their role in causing hallmark glioma symptoms, such as cognitive deficits and seizures, as well as their potential ability to drive glioma progression. Separately, mTOR signaling has been shown to have pleiotropic effects in the brain tumor microenvironment, including regulation of neuronal hyperexcitability. However, the local cellular-level effects of mTOR inhibition on glioma-induced neuronal alterations are not well understood. Here we employed neuron-specific profiling of ribosome-bound mRNA via 'RiboTag,' morphometric analysis of dendritic spines, and in vivo calcium imaging, along with pharmacological mTOR inhibition to investigate the impact of glioma burden and mTOR inhibition on these neuronal alterations. The RiboTag analysis of tumor-associated excitatory neurons showed a downregulation of transcripts encoding excitatory and inhibitory postsynaptic proteins and dendritic spine development, and an upregulation of transcripts encoding cytoskeletal proteins involved in dendritic spine turnover. Light and electron microscopy of tumor-associated excitatory neurons demonstrated marked decreases in dendritic spine density. In vivo two-photon calcium imaging in tumor-associated excitatory neurons revealed progressive alterations in neuronal activity, both at the population and single-neuron level, throughout tumor growth. This in vivo calcium imaging also revealed altered stimulus-evoked somatic calcium events, with changes in event rate, size, and temporal alignment to stimulus, which was most pronounced in neurons with high-tumor burden. A single acute dose of AZD8055, a combined mTORC1/2 inhibitor, reversed the glioma-induced alterations on the excitatory neurons, including the alterations in ribosome-bound transcripts, dendritic spine density, and stimulus evoked responses seen by calcium imaging. These results point to mTOR-driven pathological plasticity in neurons at the infiltrative margin of glioma - manifested by alterations in ribosome-bound mRNA, dendritic spine density, and stimulus-evoked neuronal activity. Collectively, our work identifies the pathological changes that tumor-associated excitatory neurons experience as both hyperlocal and reversible under the influence of mTOR inhibition, providing a foundation for developing therapies targeting neuronal signaling in glioma.
RESUMO
The mechanical stiffness of a cell's environment exerts a strong, but variable, influence on cell behavior and fate. For example, different cell types cultured on compliant substrates have opposite trends of cell migration and traction as a function of substrate stiffness. Here, we describe how a motor-clutch model of cell traction, which exhibits a maximum in traction force with respect to substrate stiffness, may provide a mechanistic basis for understanding how cells are tuned to sense the stiffness of specific microenvironments. We find that the optimal stiffness is generally more sensitive to clutch parameters than to motor parameters, but that single parameter changes are generally only effective over a small range of values. By contrast, dual parameter changes, such as coordinately increasing the numbers of both motors and clutches offer a larger dynamic range for tuning the optimum. The model exhibits distinct regimes: at high substrate stiffness, clutches quickly build force and fail (so-called frictional slippage), whereas at low substrate stiffness, clutches fail spontaneously before the motors can load the substrate appreciably (a second regime of frictional slippage). Between the two extremes, we find the maximum traction force, which occurs when the substrate load-and-fail cycle time equals the expected time for all clutches to bind. At this stiffness, clutches are used to their fullest extent, and motors are therefore resisted to their fullest extent. The analysis suggests that coordinate parameter shifts, such as increasing the numbers of motors and clutches, could underlie tumor progression and collective cell migration.
Assuntos
Movimento Celular , Microambiente Celular , Modelos Biológicos , Animais , Fenômenos Biomecânicos , Módulo de Elasticidade , HumanosRESUMO
Kinesin-5 is required for forming the bipolar spindle during mitosis. Its motor domain, which contains nucleotide and microtubule binding sites and mechanical elements to generate force, has evolved distinct properties for its spindle-based functions. In this study, we report subnanometer resolution cryoelectron microscopy reconstructions of microtubule-bound human kinesin-5 before and after nucleotide binding and combine this information with studies of the kinetics of nucleotide-induced neck linker and cover strand movement. These studies reveal coupled, nucleotide-dependent conformational changes that explain many of this motor's properties. We find that ATP binding induces a ratchet-like docking of the neck linker and simultaneous, parallel docking of the N-terminal cover strand. Loop L5, the binding site for allosteric inhibitors of kinesin-5, also undergoes a dramatic reorientation when ATP binds, suggesting that it is directly involved in controlling nucleotide binding. Our structures indicate that allosteric inhibitors of human kinesin-5, which are being developed as anti-cancer therapeutics, bind to a motor conformation that occurs in the course of normal function. However, due to evolutionarily defined sequence variations in L5, this conformation is not adopted by invertebrate kinesin-5s, explaining their resistance to drug inhibition. Together, our data reveal the precision with which the molecular mechanism of kinesin-5 motors has evolved for force generation.
Assuntos
Cinesinas/química , Cinesinas/metabolismo , Fuso Acromático/metabolismo , Sítios de Ligação , Humanos , Cinesinas/genética , Cinética , Microtúbulos/genética , Microtúbulos/metabolismo , Mitose , Modelos Moleculares , Nucleotídeos/metabolismo , Ligação Proteica , Conformação Proteica , Estrutura Terciária de Proteína , Fuso Acromático/químicaRESUMO
Glioblastoma (GBM) is a deadly brain tumor, and the kinesin motor KIF11 is an attractive therapeutic target because of its dual roles in proliferation and invasion. The clinical utility of KIF11 inhibitors has been limited by drug resistance, which has mainly been studied in animal models. We used multiplexed lineage tracing barcodes and scRNA-seq to analyze drug resistance time courses for patient-derived GBM neurospheres treated with ispinesib, a potent KIF11 inhibitor. Similar to GBM progression in patients, untreated cells lost their neural lineage identity and transitioned to a mesenchymal phenotype, which is associated with poor prognosis. In contrast, cells subjected to long-term ispinesib treatment exhibited a proneural phenotype. We generated patient-derived xenografts to show that ispinesib-resistant cells form less aggressive tumors in vivo, even in the absence of drug. Finally, we used lineage barcodes to nominate drug combination targets by retrospective analysis of ispinesib-resistant clones in the drug-naïve setting and identified drugs that are synergistic with ispinesib.
RESUMO
KIF20A is a critical kinesin for cell division and a promising anti-cancer drug target. The mechanisms underlying its cellular roles remain elusive. Interestingly, unusual coupling between the nucleotide- and microtubule-binding sites of this kinesin-6 has been reported, but little is known about how its divergent sequence leads to atypical motility properties. We present here the first high-resolution structure of its motor domain that delineates the highly unusual structural features of this motor, including a long L6 insertion that integrates into the core of the motor domain and that drastically affects allostery and ATPase activity. Together with the high-resolution cryo-electron microscopy microtubule-bound KIF20A structure that reveals the microtubule-binding interface, we dissect the peculiarities of the KIF20A sequence that influence its mechanochemistry, leading to low motility compared to other kinesins. Structural and functional insights from the KIF20A pre-power stroke conformation highlight the role of extended insertions in shaping the motor's mechanochemical cycle. Essential for force production and processivity is the length of the neck linker in kinesins. We highlight here the role of the sequence preceding the neck linker in controlling its backward docking and show that a neck linker four times longer than that in kinesin-1 is required for the activity of this motor.
Assuntos
Cinesinas , Microtúbulos , Microscopia Crioeletrônica , Cinesinas/genética , Sítios de Ligação , Divisão CelularRESUMO
Kinesin-5 crosslinks and slides apart microtubules to assemble, elongate, and maintain the mitotic spindle. Kinesin-5 is a tetramer, where two N-terminal motor domains are positioned at each end of the motor, and the coiled-coil stalk domains are organized into a tetrameric bundle through the bipolar assembly (BASS) domain. To dissect the function of the individual structural elements of the motor, we constructed a minimal kinesin-5 tetramer (mini-tetramer). We determined the x-ray structure of the extended, 34-nm BASS domain. Guided by these structural studies, we generated active bipolar kinesin-5 mini-tetramer motors from Drosophila melanogastor and human orthologues which are half the length of native kinesin-5. We then used these kinesin-5 mini-tetramers to examine the role of two unique structural adaptations of kinesin-5: 1) the length and flexibility of the tetramer, and 2) the C-terminal tails which interact with the motor domains to coordinate their ATPase activity. The C-terminal domain causes frequent pausing and clustering of kinesin-5. By comparing microtubule crosslinking and sliding by mini-tetramer and full-length kinesin-5, we find that both the length and flexibility of kinesin-5 and the C-terminal tails govern its ability to crosslink microtubules. Once crosslinked, stiffer mini-tetramers slide antiparallel microtubules more efficiently than full-length motors.