Dwell time distributions of the molecular motor myosin V.
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ABSTRACT:
SUBMITTER: Bierbaum V
PROVIDER: S-EPMC3572133 | biostudies-literature | 2013
REPOSITORIES: biostudies-literature
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Project description:Fitting dwell-time distributions with sums of exponentials is widely used to characterize histograms of open- and closed-interval durations recorded from single ion channels, as well as for other physical phenomena. However, it can be difficult to identify the contributing exponential components. Here we extend previous methods of exponential sum-fitting to present a maximum-likelihood approach that consistently detects all significant exponentials without the need for user-specified starting parameters. Instead of searching for exponentials, the fitting starts with a very large number of initial exponentials with logarithmically spaced time constants, so that none are missed. Maximum-likelihood fitting then determines the areas of all the initial exponentials keeping the time constants fixed. In an iterative manner, with refitting after each step, the analysis then removes exponentials with negligible area and combines closely spaced adjacent exponentials, until only those exponentials that make significant contributions to the dwell-time distribution remain. There is no limit on the number of significant exponentials and no starting parameters need be specified. We demonstrate fully automated detection for both experimental and simulated data, as well as for classical exponential-sum-fitting problems.
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Project description:Myosin motor domains perform an extraordinary diversity of biological functions despite sharing a common mechanochemical cycle. Motors are adapted to their function, in part, by tuning the thermodynamics and kinetics of steps in this cycle. However, it remains unclear how sequence encodes these differences, since biochemically distinct motors often have nearly indistinguishable crystal structures. We hypothesized that sequences produce distinct biochemical phenotypes by modulating the relative probabilities of an ensemble of conformations primed for different functional roles. To test this hypothesis, we modeled the distribution of conformations for 12 myosin motor domains by building Markov state models (MSMs) from an unprecedented two milliseconds of all-atom, explicit-solvent molecular dynamics simulations. Comparing motors reveals shifts in the balance between nucleotide-favorable and nucleotide-unfavorable P-loop conformations that predict experimentally measured duty ratios and ADP release rates better than sequence or individual structures. This result demonstrates the power of an ensemble perspective for interrogating sequence-function relationships.
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Project description:Neuronal dynamics result from the integration of forces developed by molecular motors, especially conventional myosins. Myosin IIC is a recently discovered nonsarcomeric conventional myosin motor, the function of which is poorly understood, particularly in relation to the separate but coupled activities of its close homologues, myosins IIA and IIB, which participate in neuronal adhesion, outgrowth and retraction. To determine myosin IIC function, we have applied a comparative functional knockdown approach by using isoform-specific antisense oligodeoxyribonucleotides to deplete expression within neuronally derived cells. Myosin IIC was found to be critical for driving neuronal process outgrowth, a function that it shares with myosin IIB. Additionally, myosin IIC modulates neuronal cell adhesion, a function that it shares with myosin IIA but not myosin IIB. Consistent with this role, myosin IIC knockdown caused a concomitant decrease in paxillin-phospho-Tyr118 immunofluorescence, similar to knockdown of myosin IIA but not myosin IIB. Myosin IIC depletion also created a distinctive phenotype with increased cell body diameter, increased vacuolization, and impaired responsiveness to triggered neurite collapse by lysophosphatidic acid. This novel combination of properties suggests that myosin IIC must participate in distinctive cellular roles and reinforces our view that closely related motor isoforms drive diverse functions within neuronal cells.
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Project description:The microtubule cytoskeleton plays important roles in cell polarity, motility and division. Microtubules inherently undergo dynamic instability, stochastically switching between phases of growth and shrinkage. In cells, some microtubule-associated proteins (MAPs) and molecular motors can further modulate microtubule dynamics. We present here the fission yeast mtr1(+), a new regulator of microtubule dynamics that appears to be not a MAP or a motor. mtr1-deletion (mtr1Δ) primarily results in longer microtubule dwell-time at the cell tip cortex, suggesting that mtr1p acts directly or indirectly as a destabilizer of microtubules. mtr1p is antagonistic to mal3p, the ortholog of mammalian EB1, which stabilizes microtubules. mal3Δ results in short microtubules, but can be partially rescued by mtr1Δ, as the double mutant mal3Δ mtr1Δ exhibits longer microtubules than mal3Δ single mutant. By sequence homology, mtr1p is predicted to be a component of the ribosomal quality control complex. Intriguingly, deletion of a predicted ribosomal gene, rps1801, also resulted in longer microtubule dwell-time similar to mtr1Δ. The double-mutant mal3Δ rps1801Δ also exhibits longer microtubules than mal3Δ single mutant alone. Our study suggests a possible involvement of mtr1p and the ribosome complex in modulating microtubule dynamics.
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Project description:Dynamic microscale strain distributions with temporal resolution of 1 s in a smooth and a cracked Ti-6Al-4V alloys during one-cycle dwell fatigue tests are illustrated in videos (URL: https://drive.google.com/drive/folders/1pit_VV2apGOpETVfaJAAtL5Xl2CNOiJ3?usp=sharing). The tensile strain distributions were measured by the video sampling moiré method from the 1-μm-pitch grid images in a scanning electron microscope. The strain concentration factors of the smooth and the cracked specimens are 1.96 and 2.65, respectively. The plastic strain increment is 0.0007 during the displacement holding time of 591s in the smoothed specimen at maximum stress of 900 MPa., and 0.0008 during the displacement holding time of 593s in the cracked specimen at maximum stress of 870 MPa. The typical strain results are analyzed in 1-s-resolved strain mapping in Ti-6Al-4V alloys during dwell fatigue in SEM by video sampling moiré [1].
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Project description:PURPOSE: To develop and test a fast ultrasonic molecular imaging technique for quantification and monitoring of angiogenesis in cancer. MATERIALS AND METHODS: A new software algorithm measuring the dwell time of contrast microbubbles in near real-time (henceforth, fast method) was developed and integrated in a clinical ultrasound system. In vivo quantification and monitoring of tumor angiogenesis during anti-VEGF antibody therapy was performed in human colon cancer xenografts in mice (n=20) using the new fast method following administration of vascular endothelial growth factor receptor 2 (VEGFR2)-targeted contrast microbubbles. Imaging results were compared with a traditional destruction/replenishment approach (henceforth, traditional method) in an intra-animal comparison. RESULTS: There was excellent correlation (R(2)=0.93; P<0.001) between the fast method and the traditional method in terms of VEGFR2-targeted in vivo ultrasonic molecular imaging with significantly higher (P=0.002) imaging signal in colon cancer xenografts using VEGFR2-targeted compared to control non-targeted contrast microbubbles. The new fast method was highly reproducible (ICC=0.87). Following anti-angiogenic therapy, ultrasonic molecular imaging signal decreased by an average of 41±10%, whereas imaging signal increased by an average of 54±8% in non-treated tumors over a 72-hour period. Decreased VEGFR2 expression levels following anti-VEGF therapy were confirmed on ex vivo immunofluorescent staining. CONCLUSIONS: Fast ultrasonic molecular imaging based on dwell time microbubble signal measurements correlates well with the traditional measurement method, and allows reliable in vivo monitoring of anti-angiogenic therapy in human colon cancer xenografts. The improved work-flow afforded by the new quantification approach may facilitate clinical translation of ultrasonic molecular imaging.
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Project description:Despite recent progress in understanding lamellipodia extension, the molecular mechanisms regulating filopodia formation remain largely unknown. Myo10 is a MyTH4-FERM myosin that localizes to the tips of filopodia and is hypothesized to function in filopodia formation. To determine whether endogenous Myo10 is required for filopodia formation, we have used scanning EM to assay the numerous filopodia normally present on the dorsal surfaces of HeLa cells. We show here that siRNA-mediated knockdown of Myo10 in HeLa cells leads to a dramatic loss of dorsal filopodia. Overexpressing the coiled coil region from Myo10 as a dominant- negative also leads to a loss of dorsal filopodia, thus providing independent evidence that Myo10 functions in filopodia formation. We also show that expressing Myo10 in COS-7 cells, a cell line that normally lacks dorsal filopodia, leads to a massive induction of dorsal filopodia. Because the dorsal filopodia induced by Myo10 are not attached to the substrate, Myo10 can promote filopodia by a mechanism that is independent of substrate attachment. Consistent with this observation, a Myo10 construct that lacks the FERM domain, the region that binds to integrin, retains the ability to induce dorsal filopodia. Deletion of the MyTH4-FERM region, however, completely abolishes Myo10's filopodia-promoting activity, as does deletion of the motor domain. Additional experiments on the mechanism of Myo10 action indicate that it acts downstream of Cdc42 and can promote filopodia in the absence of VASP proteins. Together, these data demonstrate that Myo10 is a molecular motor that functions in filopodia formation.
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