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 , Ligação de Hidrogênio , Cinesinas/metabolismo , Modelos Moleculares , Multimerização Proteica , Estrutura Quaternária de Proteína , Tubulina (Proteína)/químicaRESUMO
Kinesins and myosins hydrolyze ATP, producing force that drives spindle assembly, vesicle transport and muscle contraction. How do motors do this? Here we discuss mechanisms of motor force transduction, based on their mechanochemical cycles and conformational changes observed in crystal structures. Distortion or twisting of the central ß-sheet - proposed to trigger actin-induced Pi and ADP release by myosin, and microtubule-induced ADP release by kinesins - is shown in a movie depicting the transition between myosin ATP-like and nucleotide-free states. Structural changes in the switch I region form a tube that governs ATP hydrolysis and Pi release by the motors, explaining the essential role of switch I in hydrolysis. Comparison of the motor power strokes reveals that each stroke begins with the force-amplifying structure oriented opposite to the direction of rotation or swing. Motors undergo changes in their mechanochemical cycles in response to small-molecule inhibitors, several of which bind to kinesins by induced fit, trapping the motors in a state that resembles a force-producing conformation. An unusual motor activator specifically increases mechanical output by cardiac myosin, potentially providing valuable information about its mechanism of function. Further study is essential to understand motor mechanochemical coupling and energy transduction, and could lead to new therapies to treat human disease.
Assuntos
Proteínas do Citoesqueleto/metabolismo , Cinesinas/metabolismo , Miosinas/metabolismo , Trifosfato de Adenosina/metabolismo , Animais , Fenômenos Biomecânicos , Humanos , Modelos Biológicos , Proteínas Motores Moleculares/metabolismoRESUMO
A festive group of â¼150 current and former students, postdoctoral and other associates, and colleagues gathered during the weekend of April 12-14, 2013 to celebrate Joe Gall's 85th birthday. The gathering, hosted by the Carnegie Institution for Science, Department of Embryology (Allan Spradling, Director) and organized by a group of Joe's current and former students (Zehra Nizami, Alison Singer, Ji-Long Liu, Virginia Zakian, Susan Gerbi), was held in Baltimore, MD. Dinners and symposia extending over 3 days celebrated Joe's scientific findings over the years, together with those of his former students, postdoctoral fellows, and other associates (see program at https://sites.google.com/site/gallsymposium2013/ ).
Assuntos
Cromossomos/genética , Congressos como Assunto , DNA Ribossômico/isolamento & purificação , DNA Ribossômico/genética , História do Século XX , História do Século XXI , Sociedades CientíficasRESUMO
Kinesin motor proteins hydrolyze ATP to produce force for spindle assembly and vesicle transport, performing essential functions in cell division and motility, but the structural changes required for force generation are uncertain. We now report high-resolution structures showing new transitions in the kinesin mechanochemical cycle, including power stroke fluctuations upon ATP binding and a post-hydrolysis state with bound ADP + free phosphate. We find that rate-limiting ADP release occurs upon microtubule binding, accompanied by central ß-sheet twisting, which triggers the power stroke - stalk rotation and neck mimic docking - upon ATP binding. Microtubule release occurs with ß-strand-to-loop transitions, implying that ß-strand refolding induces Pi release and the recovery stroke. The strained ß-sheet during the power stroke and strand-to-loop transitions identify the ß-sheet as the long-sought motor spring.
RESUMO
BACKGROUND: Anastral spindles assemble by a mechanism that involves microtubule nucleation and growth from chromatin. It is still uncertain whether γ-tubulin, a microtubule nucleator essential for mitotic spindle assembly and maintenance, plays a role. Not only is the requirement for γ-tubulin to form anastral Drosophila oocyte meiosis I spindles controversial, but its presence in oocyte meiosis I spindles has not been demonstrated and is uncertain. RESULTS: We show, for the first time, using a bright GFP fusion protein and live imaging, that the Drosophila maternally-expressed γTub37C is present at low levels in oocyte meiosis I spindles. Despite this, we find that formation of bipolar meiosis I spindles does not require functional γTub37C, extending previous findings by others. Fluorescence photobleaching assays show rapid recovery of γTub37C in the meiosis I spindle, similar to the cytoplasm, indicating weak binding by γTub37C to spindles, and fits of a new, potentially more accurate model for fluorescence recovery yield kinetic parameters consistent with transient, diffusional binding. CONCLUSIONS: The FRAP results, together with its mutant effects late in meiosis I, indicate that γTub37C may perform a role subsequent to metaphase I, rather than nucleating microtubules for meiosis I spindle formation. Weak binding to the meiosis I spindle could stabilize pre-existing microtubules or position γ-tubulin for function during meiosis II spindle assembly, which follows rapidly upon oocyte activation and completion of the meiosis I division.
Assuntos
Drosophila/metabolismo , Fuso Acromático/fisiologia , Tubulina (Proteína)/metabolismo , Animais , Drosophila/embriologia , Feminino , Meiose , Metáfase , Oócitos/metabolismo , Fotodegradação , Tubulina (Proteína)/genéticaRESUMO
Analysis of a mutant with altered directionality has led to new insights into motor directionality. The prediction from current models for processivity of a two-heads-bound state has been confirmed by electron microscopy for myosin V and by unbinding experiments for kinesin. Evidence is emerging that non-processive motors bind their filament with one head, hydrolyze ATP and then release, requiring binding by a second motor to complete a step.
Assuntos
Proteínas Motores Moleculares/metabolismo , Animais , Citoesqueleto/metabolismo , Cinesinas/química , Cinesinas/metabolismo , Microtúbulos/metabolismo , Modelos Biológicos , Modelos Moleculares , Proteínas Motores Moleculares/química , Proteínas Motores Moleculares/genética , Mutação , Miosinas/química , Miosinas/metabolismoRESUMO
BACKGROUND: Kinesin motors hydrolyze ATP to produce force and move along microtubules, converting chemical energy into work by a mechanism that is only poorly understood. Key transitions and intermediate states in the process are still structurally uncharacterized, and remain outstanding questions in the field. Perturbing the motor by introducing point mutations could stabilize transitional or unstable states, providing critical information about these rarer states. RESULTS: Here we show that mutation of a single residue in the kinesin-14 Ncd causes the motor to release ADP and hydrolyze ATP faster than wild type, but move more slowly along microtubules in gliding assays, uncoupling nucleotide hydrolysis from force generation. A crystal structure of the motor shows a large rotation of the stalk, a conformation representing a force-producing stroke of Ncd. Three C-terminal residues of Ncd, visible for the first time, interact with the central beta-sheet and dock onto the motor core, forming a structure resembling the kinesin-1 neck linker, which has been proposed to be the primary force-generating mechanical element of kinesin-1. CONCLUSIONS: Force generation by minus-end Ncd involves docking of the C-terminus, which forms a structure resembling the kinesin-1 neck linker. The mechanism by which the plus- and minus-end motors produce force to move to opposite ends of the microtubule appears to involve the same conformational changes, but distinct structural linkers. Unstable ADP binding may destabilize the motor-ADP state, triggering Ncd stalk rotation and C-terminus docking, producing a working stroke of the motor.
Assuntos
Proteínas de Drosophila/química , Proteínas de Drosophila/metabolismo , Cinesinas/química , Cinesinas/metabolismo , Difosfato de Adenosina/metabolismo , Trifosfato de Adenosina/metabolismo , Sequência de Aminoácidos , Substituição de Aminoácidos , Animais , Sítios de Ligação , Cristalografia por Raios X , Proteínas de Drosophila/genética , Drosophila melanogaster , Hidrólise , Cinesinas/genética , Cinética , Microtúbulos/metabolismo , Modelos Moleculares , Mutação , Estrutura Secundária de Proteína , RotaçãoRESUMO
Assembly of an anastral spindle was modeled as a two-stage process: first, the aggregation of microtubule foci or asters around the chromosomes, and second, the elongation of cross-linked microtubules and onset of bipolarity. Several possibilities involving diffusion and transport were investigated for the first stage, and the most feasible was found to be binding of the asters to cytoskeletal filaments and directed transport toward the chromosomes. For the second stage, a differential-equation model was formulated and solved numerically; it involves cross-linking of microtubules with those aligned with the spindle axis and between microtubules bound to different chromosomes, and sliding of microtubules along the spindle axis to elongate the spindle. Ncd was postulated to perform both functions. The model shows that spindle formation begins with rapid cross-linking of microtubules, followed by elongation, which continues until the population of microtubules aligned with the spindle axis is depleted and microtubules cross-linking different chromosomes dominate. It also shows that when sliding is inhibited, short bipolar spindles still form, and if clustering is enhanced, normal-length spindles can form, although requiring longer assembly time. These findings are consistent with spindle assembly in live wild-type and ncd mutant Drosophila oocytes.
Assuntos
Cromossomos/metabolismo , Microtúbulos/metabolismo , Modelos Biológicos , Modelos Genéticos , Algoritmos , Animais , Animais Geneticamente Modificados , Transporte Biológico/fisiologia , Simulação por Computador , Citoesqueleto/metabolismo , Difusão , Drosophila , Proteínas de Drosophila/genética , Cinesinas/genética , Cinética , Meiose/fisiologia , Oócitos/fisiologiaRESUMO
In recent years the kinesin superfamily has become so large that several different naming schemes have emerged, leading to confusion and miscommunication. Here, we set forth a standardized kinesin nomenclature based on 14 family designations. The scheme unifies all previous phylogenies and nomenclature proposals, while allowing individual sequence names to remain the same, and for expansion to occur as new sequences are discovered.
Assuntos
Cinesinas/química , Cinesinas/classificação , Terminologia como Assunto , Animais , Humanos , Família MultigênicaRESUMO
The mechanism by which motor proteins hydrolyze ATP and move along cytoskeletal filaments is still unknown. One approach to deciphering the mechanism is to correlate steps of ATP hydrolysis with structural states of the motors to determine the changes the motors undergo during the hydrolysis cycle. Unfortunately, available crystal structures represent only a few steps of the cycle and obtaining atomic structures that represent the motors bound to their filament has been difficult. Now, two new myosin crystal structures have been reported that show features expected for myosin motors bound in rigor to actin. The two new structures show changes at both the actin-binding surface and the active site that have not been observed previously.
Assuntos
Miosinas/química , Actinas/fisiologia , Trifosfato de Adenosina/metabolismo , Hidrólise , Cinesinas/química , Cinesinas/fisiologia , Modelos Biológicos , Modelos Moleculares , Proteínas Motores Moleculares , Miosinas/fisiologia , Ligação Proteica , Conformação ProteicaRESUMO
Brain stem cells stop dividing in late Drosophila embryos and begin dividing again in early larvae after feeding induces reactivation. Quiescent neural stem cells (qNSCs) display an unusual cytoplasmic protrusion that is no longer present in reactivated NSCs. The protrusions join the qNSCs to the neuropil, brain regions that are thought to maintain NSCs in an undifferentiated state, but the function of the protrusions is not known. Here we show that qNSC protrusions contain clustered mitochondria that are likely maintained in position by slow forward-and-backward microtubule growth. Larvae treated with a microtubule-stabilizing drug show bundled microtubules and enhanced mitochondrial clustering in NSCs, together with reduced qNSC reactivation. We further show that intestinal stem cells contain mitochondria-enriched protrusions. The qNSC and intestinal stem-cell protrusions differ from previously reported cytoplasmic extensions by forming stem-cell-to-niche mitochondrial bridges that could potentially both silence genes and sense signals from the stem cell niche.
Assuntos
Células-Tronco Adultas/metabolismo , Encéfalo/citologia , Encéfalo/metabolismo , Extensões da Superfície Celular/metabolismo , Mucosa Intestinal/citologia , Mucosa Intestinal/metabolismo , Mitocôndrias/metabolismo , Células-Tronco Adultas/ultraestrutura , Animais , Extensões da Superfície Celular/ultraestrutura , Drosophila , Imunofluorescência , Microscopia de Fluorescência , Microtúbulos/metabolismoRESUMO
Fluorescence recovery after photobleaching has been widely used to study dynamic processes in the cell, but less frequently to analyze binding interactions and extract binding constants. Here we use it to analyze gamma-tubulin binding to the mitotic spindle and centrosomes to determine the role of gamma-tubulin in microtubule nucleation in the spindle. We find rapid gamma-tubulin turnover in mitotic spindles of Drosophila early embryos, characterized by diffusional interactions and weak binding, differing from centrosomes with tight binding interactions. The diffusion coefficient of gamma-tubulin is consistent with a major species existing in the cytoplasm as the less efficiently nucleating gamma-tubulin small complex (gammaTuSC) or gamma-tubulin, rather than gamma-tubulin ring complex (gammaTuRC). The fluorescence recovery kinetics we observe implies that gamma-tubulin functions by binding weakly to spindle microtubules. gamma-Tubulin may interact transiently with the spindle, nucleating microtubules very rapidly, differing from centrosomes, where gamma-tubulin binds tightly to nucleate microtubules.
Assuntos
Fuso Acromático/metabolismo , Tubulina (Proteína)/metabolismo , Animais , Centrossomo/metabolismo , Difusão , Drosophila/citologia , Drosophila/metabolismo , Recuperação de Fluorescência Após Fotodegradação , Cinética , Microtúbulos/metabolismo , Ligação Proteica , Fatores de TempoAssuntos
Cinesinas/classificação , Cinesinas/metabolismo , Animais , Transporte Biológico/genética , Transporte Biológico/fisiologia , Humanos , Cinesinas/genética , Meiose/genética , Meiose/fisiologia , Mitose/genética , Mitose/fisiologia , Proteínas Motores Moleculares/classificação , Proteínas Motores Moleculares/genética , Proteínas Motores Moleculares/metabolismoRESUMO
Kinesin microtubule motor proteins play essential roles in division, including attaching chromosomes to spindles and crosslinking microtubules for spindle assembly. Human kinesin-14 KIFC1 is unique in that cancer cells with amplified centrosomes are dependent on the motor for viable division because of its ability to cluster centrosomes and form bipolar spindles, but it is not required for division in almost all normal cells. Screens for small molecule inhibitors of KIFC1 have yielded several candidates for further development, but obtaining structural data to determine their sites of binding has been difficult. Here we compare a previously unreported KIFC1 crystal structure with new structures of two closely related kinesin-14 proteins, Ncd and KIFC3, to determine the potential binding site of a known KIFC1 ATPase inhibitor, AZ82. We analyze the previously identified kinesin inhibitor binding sites and identify features of AZ82 that favor binding to one of the sites, the α4/α6 site. This selectivity can be explained by unique structural features of the KIFC1 α4/α6 binding site. These features may help improve the drug-like properties of AZ82 and other specific KIFC1 inhibitors.
Assuntos
Alanina/análogos & derivados , Inibidores Enzimáticos/química , Inibidores Enzimáticos/metabolismo , Cinesinas/antagonistas & inibidores , Cinesinas/química , Piridinas/química , Piridinas/metabolismo , Alanina/química , Alanina/metabolismo , Sítios de Ligação , Cristalografia por Raios X , Proteínas de Drosophila/química , Proteínas de Drosophila/metabolismo , Humanos , Cinesinas/metabolismo , Modelos Moleculares , Ligação Proteica , Conformação ProteicaRESUMO
Kinesin-13 motors are unusual in that they do not walk along microtubules, but instead diffuse to the ends, where they remove tubulin dimers, regulating microtubule dynamics. Here we show that Drosophila kinesin-13 klp10A regulates oocyte meiosis I spindle length and is haplo-insufficient - KLP10A, reduced by RNAi or a loss-of-function P element insertion mutant, results in elongated and mispositioned oocyte spindles, and abnormal cortical microtubule asters and aggregates. KLP10A knockdown by RNAi does not significantly affect microtubule growth rates in oocyte spindles, but, unexpectedly, EB1 binding and unbinding are slowed, suggesting a previously unobserved role for kinesin-13 in mediating EB1 binding interactions with microtubules. Kinesin-13 may regulate spindle length both by disassembling subunits from microtubule ends and facilitating EB1 binding to plus ends. We also observe an increased number of paused microtubules in klp10A RNAi knockdown spindles, consistent with a reduced frequency of microtubule catastrophes. Overall, our findings indicate that reduced kinesin-13 decreases microtubule disassembly rates and affects EB1 interactions with microtubules, rather than altering microtubule growth rates, causing spindles to elongate and abnormal cortical microtubule asters and aggregates to form.
RESUMO
Kinesin motors hydrolyze ATP to produce force and do work in the cell--how the motors do this is not fully understood, but is thought to depend on the coupling of ATP hydrolysis to microtubule binding by the motor. Transmittal of conformational changes from the microtubule- to the nucleotide-binding site has been proposed to involve the central ß-sheet, which could undergo large structural changes important for force production. We show here that mutation of an invariant residue in loop L7 of the central ß-sheet of the Drosophila kinesin-14 Ncd motor alters both nucleotide and microtubule binding, although the mutated residue is not present in either site. Mutants show weak-ADP/tight-microtubule binding, instead of tight-ADP/weak-microtubule binding like wild type--they hydrolyze ATP faster than wild type, move faster in motility assays, and assemble long spindles with greatly elongated poles, which are also produced by simulations of assembly with tighter microtubule binding and faster sliding. The mutated residue acts like a mechanochemical coupling element--it transmits changes between the microtubule-binding and active sites, and can switch the state of the motor, increasing mechanical output by the motor. One possibility, based on our findings, is that movements by the residue and the loop that contains it could bend or distort the central ß-sheet, mediating free energy changes that lead to force production.
Assuntos
Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Drosophila/genética , Drosophila/metabolismo , Cinesinas/genética , Cinesinas/metabolismo , Microtúbulos/metabolismo , Nucleotídeos/metabolismo , Trifosfato de Adenosina/metabolismo , Animais , Domínio Catalítico , Drosophila/química , Proteínas de Drosophila/química , Cinesinas/química , Modelos Moleculares , Mutação Puntual , Ligação Proteica , Estrutura Secundária de ProteínaRESUMO
Rotation of the coiled-coil stalk of the kinesin-14 motors is thought to drive displacements or steps by the motor along microtubules, but the structural changes that trigger stalk rotation and the nucleotide state in which it occurs are not certain. Here we report a kinesin-14 neck mutant that releases ADP more slowly than wild type and shows weaker microtubule affinity, consistent with defective stalk rotation. Unexpectedly, crystal structures show the stalk fully rotated - neck-motor interactions destabilize the stalk, causing it to rotate and ADP to be released, and alter motor affinity for microtubules. A new structural pathway accounts for the coupling of stalk rotation - the force-producing stroke - to changes in motor affinity for nucleotide and microtubules. Sequential disruption of salt bridges that stabilize the unrotated stalk could cause the stalk to initiate and complete rotation in different nucleotide states.
Assuntos
Cinesinas/fisiologia , Cristalografia por Raios X , Cinesinas/química , Cinesinas/metabolismo , Cinética , Microtúbulos/metabolismo , Modelos MolecularesRESUMO
The nonprocessive kinesin-14 Ncd motor binds to microtubules and hydrolyzes ATP, undergoing a single displacement before releasing the microtubule. A lever-like rotation of the coiled-coil stalk is thought to drive Ncd displacements or steps along microtubules. Crystal structures and cryoelectron microscopy reconstructions imply that stalk rotation is correlated with ADP release and microtubule binding by the motor. Here we report FRET assays showing that the end of the stalk is more than ~9nm from the microtubule when wild-type Ncd binds microtubules without added nucleotide, but the stalk is within ~6nm of the microtubule surface when the microtubule-bound motor binds an ATP analogue, matching the rotated state observed in crystal structures. We propose that the stalk rotation is initiated when the motor binds to microtubules and releases ADP, and is completed when ATP binds.
Assuntos
Cinesinas/química , Difosfato de Adenosina/metabolismo , Substituição de Aminoácidos , Transferência Ressonante de Energia de Fluorescência , Cinesinas/genética , Cinesinas/metabolismo , Microtúbulos/química , Modelos Teóricos , Mutação , Ligação Proteica , Estrutura Terciária de Proteína , RotaçãoRESUMO
The kinesin-13 motor, KLP10A, destabilizes microtubules at their minus ends in mitosis and binds to polymerizing plus ends in interphase, regulating spindle and microtubule dynamics. Little is known about kinesin-13 motors in meiosis. In this study, we report that KLP10A localizes to the unusual pole bodies of anastral Drosophila melanogaster oocyte meiosis I spindles as well as spindle fibers, centromeres, and cortical microtubules. We frequently observe the pole bodies attached to cortical microtubules, indicating that KLP10A could mediate spindle anchoring to the cortex via cortical microtubules. Oocytes treated with drugs that suppress microtubule dynamics exhibit spindles that are reoriented more vertically to the cortex than untreated controls. A dominant-negative klp10A mutant shows both reoriented and shorter oocyte spindles, implying that, unexpectedly, KLP10A may stabilize rather than destabilize microtubules, regulating spindle length and positioning the oocyte spindle. By altering microtubule dynamics, KLP10A could promote spindle reorientation upon oocyte activation.
Assuntos
Proteínas de Drosophila/metabolismo , Cinesinas/metabolismo , Meiose/fisiologia , Microtúbulos/metabolismo , Proteínas Motores Moleculares/metabolismo , Oócitos/metabolismo , Fuso Acromático/metabolismo , Trifosfato de Adenosina/metabolismo , Animais , Movimento Celular/genética , Polaridade Celular/genética , Células Cultivadas , Proteínas de Drosophila/genética , Drosophila melanogaster/metabolismo , Drosophila melanogaster/ultraestrutura , Feminino , Regulação da Expressão Gênica no Desenvolvimento/genética , Cinesinas/genética , Meiose/genética , Microtúbulos/ultraestrutura , Proteínas Motores Moleculares/genética , Mutação/genética , Oócitos/ultraestrutura , Ligação Proteica/fisiologia , Fuso Acromático/ultraestruturaRESUMO
The Ncd kinesin-14 motor is required for meiotic spindle assembly in Drosophila oocytes and produces force in mitotic spindles that opposes other motors. Despite extensive studies, the way the motor binds to the spindle to perform its functions is not well understood. By analyzing Ncd deleted for the conserved head or the positively charged tail, we found that the tail is essential for binding to spindles and centrosomes, but both the head and tail are needed for normal spindle assembly and function. Fluorescence photobleaching assays to analyze binding interactions with the spindle yielded data for headless and full-length Ncd that did not fit well to previous recovery models. We report a new model that accounts for Ncd transport towards the equator revealed by fluorescence flow analysis of early mitotic spindles and gives rate constants that confirm the dominant role the Ncd tail plays in binding to the spindle. By contrast, the head binds weakly to spindles based on analysis of the tailless fluorescence recovery data. Minus-end Ncd thus binds tightly to spindles and is transported in early metaphase towards microtubule plus-ends, the opposite direction to that in which the motor moves, to produce force in the spindle later in mitosis.