Understanding the role of transport velocity in biomotor-powered microtubule spool assembly

We examined the sensitivity of microtubule spools to transport velocity. Perhaps surprisingly, we determined that the steady-state number and size of spools remained constant over a seven-fold range of velocities. Our data on the kinetics of spool assembly further suggest that the main mechanisms underlying spool growth vary during assembly

Membrane mediated motor kinetics in microtubule gliding assays.

Motor-based transport mechanisms are critical for a wide range of eukaryotic cell functions, including the transport of vesicle cargos over long distances. Our understanding of the factors that control and regulate motors when bound to a lipid substrate is however incomplete. We used microtubule gliding assays on a lipid bilayer substrate to investigate the role of membrane diffusion in kinesin-1 on/off binding kinetics and thereby transport velocity. Fluorescence imaging experiments demonstrate motor clustering on single microtubules due to membrane diffusion in the absence of ATP, followed by rapid ATP-induced dissociation during gliding. Our experimental data combined with analytical modeling show that the on/off binding kinetics of the motors are impacted by diffusion and, as a consequence, both the effective binding and unbinding rates for motors are much lower than the expected bare rates. Our results suggest that motor diffusion in the membrane can play a significant role in transport by impacting motor kinetics and can therefore function as a regulator of intracellular transport dynamics

Optimizing Protein–Protein van der Waals Interactions for the AMBER ff9x/ff12 Force Field

The quality of molecular dynamics (MD) simulations relies heavily on the accuracy of the underlying force field. In recent years, considerable effort has been put into developing more accurate dihedral angle potentials for MD force fields, but relatively little work has focused on the nonbonded parameters, many of which are two decades old. In this work, we assess the accuracy of protein–protein van der Waals interactions in the AMBER ff9x/ff12 force field. Across a test set of 44 neat organic liquids containing the moieties present in proteins, we find root-mean-square (RMS) errors of 1.26 kcal/mol in enthalpy of vaporization and 0.36 g/cm³ in liquid densities. We then optimize the van der Waals radii and well depths for all of the relevant atom types using these observables, which lowers the RMS errors in enthalpy of vaporization and liquid density of our validation set to 0.59 kcal/mol (53% reduction) and 0.019 g/cm³ (46% reduction), respectively. Limitations in our parameter optimization were evident for certain atom types, however, and we discuss the implications of these observations for future force field development

Heterogeneity in kinesin function

The kinesin family proteins are often studied as prototypical molecular motors; a deeper understanding of them can illuminate regulation of intracellular transport. It is typically assumed that they function identically. Here we find that this assumption of homogeneous function appears incorrect: variation among motors’ velocities in vivo and in vitro is larger than the stochastic variation expected for an ensemble of “identical” motors. When moving on microtubules, slow and fast motors are persistently slow, and fast, respectively. We develop theory that provides quantitative criteria to determine whether the observed single-molecule variation is too large to be generated from an ensemble of identical molecules. To analyze such heterogeneity, we group traces into homogeneous sub-ensembles. Motility studies varying the temperature, pH and glycerol concentration suggest at least 2 distinct functional states that are independently affected by external conditions. We end by investigating the functional ramifications of such heterogeneity through Monte-Carlo multi-motor simulations

Heterogeneity in kinesin function

The kinesin family proteins are often studied as prototypical molecular motors; a deeper understanding of them can illuminate regulation of intracellular transport. It is typically assumed that they function identically. Here we find that this assumption of homogeneous function appears incorrect: variation among motors’ velocities in vivo and in vitro is larger than the stochastic variation expected for an ensemble of “identical” motors. When moving on microtubules, slow and fast motors are persistently slow, and fast, respectively. We develop theory that provides quantitative criteria to determine whether the observed single-molecule variation is too large to be generated from an ensemble of identical molecules. To analyze such heterogeneity, we group traces into homogeneous sub-ensembles. Motility studies varying the temperature, pH and glycerol concentration suggest at least 2 distinct functional states that are independently affected by external conditions. We end by investigating the functional ramifications of such heterogeneity through Monte-Carlo multi-motor simulations

Regulation of in vivo dynein force production by CDK5 and 14-3-3ε and KIAA0528.

Single-molecule cytoplasmic dynein function is well understood, but there are major gaps in mechanistic understanding of cellular dynein regulation. We reported a mode of dynein regulation, force adaptation, where lipid droplets adapt to opposition to motion by increasing the duration and magnitude of force production, and found LIS1 and NudEL to be essential. Adaptation reflects increasing NudEL-LIS1 utilization; here, we hypothesize that such increasing utilization reflects CDK5-mediated NudEL phosphorylation, which increases the dynein-NudEL interaction, and makes force adaptation possible. We report that CDK5, 14-3-3ε, and CDK5 cofactor KIAA0528 together promote NudEL phosphorylation and are essential for force adaptation. By studying the process in COS-1 cells lacking Tau, we avoid confounding neuronal effects of CDK5 on microtubules. Finally, we extend this in vivo regulatory pathway to lysosomes and mitochondria. Ultimately, we show that dynein force adaptation can control the severity of lysosomal tug-of-wars among other intracellular transport functions involving high force

From Cancer Mimicking Orphan Lung Disease to Orphan Thoracic Oncology

International audienceA variety of rare neoplastic and non-neoplastic disorders may develop in the lung, the pleura, and the mediastinum. Some may have a propensity to mimic lung carcinoma as well as benign orphan lung diseases at some level of examination, as they may share with these clinical, imaging, pathological, and even molecular features. Challenges in the differential diagnoses among reciprocal mimics are well illustrated through examples as bronchioloalveolar carcinoma, primary pulmonary lymphomas, and vascular sarcomas. Pseudotumors have further been described, actually corresponding to a heterogeneous group of diseases characterised by circumscribed fibrous tissue and inflammatory cells. Among the inflammatory pseudotumors, neoplastic/non-neoplastic borderline disorders have been identified, such as inflammatory myofibroblastic tumor, which presents with clonal proliferation and has eventually emerged as a true neoplasm. Finally, some rare pulmonary diseases are emerging as borderline neoplastic non-neoplastic disorders, that require multidisciplinary expertise both in the field of orphan pulmonary diseases and in thoracic oncology, including amyloidosis or even Langerhans cell histiocytosis. Ultimately, implementing multi-disciplinary expert consensus is mandatory to determine the optimal management of these disorders