Structural and Functional Characterization of a Novel Heterodimeric Kinesin in Candida albicans

Kinesins are molecular motors that transport intracellular cargos along microtubules (MTs) and influence the organization and dynamics of the MT cytoskeleton. Their force-generating functions arise from conformational changes in their motor domain as ATP is bound and hydrolyzed, and products are released. In the budding yeast Saccharomyces cerevisiae, the Kar3 kinesin forms heterodimers with one of two non-catalytic kinesin-like proteins, Cik1 and Vik1, which lack the ability to bind ATP, and yet they retain the capacity to bind MTs. Cik1 and Vik1 also influence and respond to the MT-binding and nucleotide states of Kar3, and differentially regulate the functions of Kar3 during yeast mating and mitosis. The mechanism by which Kar3/Cik1 and Kar3/Vik1 dimers operate remains unknown, but has important implications for understanding mechanical coordination between subunits of motor complexes that traverse cytoskeletal tracks. In this study, we show that the opportunistic human fungal pathogen Candida albicans (Ca) harbors a single version of this unique form of heterodimeric kinesin and we present the first in vitro characterization of this motor. Like its budding yeast counterpart, the Vik1-like subunit binds directly to MTs and strengthens the MT-binding affinity of the heterodimer. However, in contrast to ScKar3/Cik1 and ScKar3/Vik1, CaKar3/Vik1 exhibits weaker overall MT-binding affinity and lower ATPase activity. Preliminary investigations using a multiple motor motility assay indicate CaKar3/Vik1 may not be motile. Using a maltose binding protein tagging system, we determined the X-ray crystal structure of the CaKar3 motor domain and observed notable differences in its nucleotide-binding pocket relative to ScKar3 that appear to represent a previously unobserved state of the active site. Together, these studies broaden our knowledge of novel kinesin motor assemblies and shed new light on structurally dynamic regions of Kar3/Vik1-like motor complexes that help mediate mechanical coordination of its subunits.

Characterization and localization of dynein and myosins V and VI in the ovaries of queen bees

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Immunolocalization of myosin Va in the developing nervous system of embryonic chicks

Myosins are molecular motors associated with the actin cytoskeleton that participate in the mechanisms of cellular motility. During the development of the nervous system, migration of nerve cells to specific sites, extension of growth cones, and axonal transport are dramatic manifestations of cellular motility. We demonstrate, via immunoblots, the expression of myosin Va during early stages of embryonic development in chicks, extending from the blastocyst period to the beginning of the fetal period. The expression of myosin Va in specific regions and cellular structures of the nervous system during these early stages was determined by immunocytochemistry using a polyclonal antibody. Whole mounts of chick embryos at 24-30-h stages showed intense immunoreactivity of the neural tube in formation along its full extent. Cross-sections at these stages of development showed strong labeling in neuroepithelial cells at the basal and apical regions of the neural tube wall. Embryos at more advanced periods of development (48h and 72 h) showed distinctive immunolabeling of neuroepithelial cells, neuroblasts and their cytoplasmic extensions in the mantle layer of the stratified neural tube wall, and neuroblasts and their cytoplasmic extensions in the internal wall of the optic cup, as well as a striking labeling of cells in the apparent nuclei of cranial nerves and budding fibers. These immunolocalization studies indicate temporal and site-specific expression of myosin Va during chick embryo development, suggesting that myosin Va expression is related to recruitment for specific cellular tasks.

Development of a recombinant fusion protein based on the dynein light chain LC8 for non-viral gene delivery

The low efficiency of gene transfer is a recurrent problem in DNA vaccine development and gene therapy studies using non-viral vectors such as plasmid DNA (pDNA). This is mainly due to the fact that during their traffic to the target cell's nuclei, plasmid vectors must overcome a series of physical, enzymatic and diffusional barriers. The main objective of this work is the development of recombinant proteins specifically designed for pDNA delivery, which take advantage of molecular motors like dynein, for the transport of cargos from the periphery to the centrosome of mammalian cells. A DNA binding sequence was fused to the N-terminus of the recombinant human dynein light chain LC8. Expression studies indicated that the fusion protein was correctly expressed in soluble form using E. coli BL21(DE3) strain. As expected, gel permeation assays found the purified protein mainly present as dimers, the functional oligomeric state of LC8. Gel retardation assays and atomic force microscopy proved the ability of the fusion protein to interact and condense pDNA. Zeta potential measurements indicated that LC8 with DNA binding domain (LD4) has an enhanced capacity to interact and condense pDNA, generating positively charged complexes. Transfection of cultured HeLa cells confirmed the ability of the LD4 to facilitate pDNA uptake and indicate the involvement of the retrograde transport in the intracellular trafficking of pDNA: LD4 complexes. Finally, cytotoxicity studies demonstrated a very low toxicity of the fusion protein vector, indicating the potential for in vivo applications. The study presented here is part of an effort to develop new modular shuttle proteins able to take advantage of strategies used by viruses to infect mammalian cells, aiming to provide new tools for gene therapy and DNA vaccination studies. (C) 2012 Elsevier B.V. All rights reserved.

Anomalous diffusion evidenced by particle tracking at high frame rates.

Diffusion is a common phenomenon in nature and generally is associated with a system trying to reach a local or a global equilibrium state, as a result of highly irregular individual particle motion. Therefore it is of fundamental importance in physics, chemistry and biology. Particle tracking in complex fluids can reveal important characteristics of its properties. In living cells, we coat the microbead with a peptide (RGD) that binds to integrin receptors at the plasma membrane, which connects to the CSK. This procedure is based on the hypothesis that the microsphere can move only if the structure where it is attached move as well. Then, the observed trajectory of microbeads is a probe of the cytoskeleton (CSK), which is governed by several factors, including thermal diffusion, pressure gradients, and molecular motors. The possibility of separating the trajectories into passive and active diffusion may give information about the viscoelasticity of the cell structure and molecular motors activity. And also we could analyze the motion via generalized Stokes-Einstein relation, avoiding the use of any active techniques. Usually a 12 to 16 Frames Per Second (FPS) system is used to track the microbeads in cell for about 5 minutes. Several factors make this FPS limitation: camera computer communication, light, computer speed for online analysis among others. Here we used a high quality camera and our own software, developed in C++ and Linux, to reach high FPS. Measurements were conducted with samples for 10£ and 20£ objectives. We performed sequentially images with different intervals, all with 2 ¹s exposure. The sequences of intervals are in milliseconds: 4 5 ms (maximum speed) 14, 25, 50 and 100 FPS. Our preliminary results highlight the difference between passive and active diffusion, since the passive diffusion is represented by a Gaussian in the distribution of displacements of the center of mass of individual beads between consecutive frames. However, the active process, or anomalous diffusion, shows as long tails in the distribution of displacements.

Responsive Bürstenpolymere

'Responsive' Bürstenpolymere Bürstenpolymere sind definiert verzweigte Makromoleküle, die aus einer Hauptkette und vielen darauf (kovalent) gepfropften Seitenketten bestehen; ist der Pfropfungsgrad hoch und die Hauptkette wesentlich länger als die Seitenketten, dann haben sie die Form semiflexibler molekularer Zylinder. Lassen sich Form bzw. Ausdehnung eines solchen Zylinders gezielt ansteuern, dann könnten diese Moleküle entweder als (Nano-)Sensoren für die entsprechende Umgebungsbedingung oder als molekulare Motoren eingesetzt werden. Die Idee responsiver Bürstenpolymere beruht auf folgender Überlegung: Die gestreckte Konformation der Hauptkette ist entropisch gegenüber einem entsprechenden Knäuel benachteiligt, weshalb sie ,molekulare Federn‘ darstellen, die auf Änderung der repulsiven Wechselwirkung zwischen den Seitenketten reagieren. Dies wurde für den Wechsel zwischen gutem und schlechtem Lösungsmitteln untersucht. Ein zweites Konzept zur Änderung der Molekülform beruht auf der intramolekularen Phasentrennung (,Segmentbildung‘) miteinander unverträglicher Seitenketten in selektiven Lösungsmitteln, da die Hauptkette durch Ausbildung von Mikrophasen entlang des Moleküls ebenfalls aus ihrer gestreckten Form gebracht werden sollte. Die dritte Möglichkeit zur Änderung der Konformation ist die intramolekulare Vernetzung von Seitenketten, die ebenfalls zu verringerter Abstoßung und damit zur Verkürzung der Zylinder führen sollte. Eine weitere wichtige Untersuchung der Arbeit war der Übergang einer geknäuelten Hauptkette zu einer gestreckten Bürste als Funktion der Pfropfdichte. Zur Beantwortung dieser Fragestellungen wurden zylindrische Bürstenpolymere durch ,Grafting Trough‘ und ,Grafting Onto‘ synthetisiert (PS bzw. PI/PS und PnBMA/PMAA mit Kern/Schale- und ,Segment‘-Architektur) und systematisch Pfropfdichte, Vernetzungsgrad (Vernetzung durch gamma-Bestrahlung) und Lösungsbedingungen verändert. Die Möglichkeit gezielter Ansteuerung der Konformationsänderung durch Vernetzung konnte nach polymeranaloger Modifikation von PI/PS-Bürstenpolymeren durch Photovernetzung und vernetzende Komplexierung erfolgreich bestätigt werden. Zur Untersuchung der Probenreihen wurden AFM, Licht- und Neutronenstreuung herangezogen. Die Analysen bestätigten konsistent die Änderung von Steifigkeit, Zylinderquerschnitt und Streckung der Hauptkette durch Variation von Pfropfdichte, Vernetzung und Lösungsmittelqualität. Für die Änderung der Pfropfdichte gehorchen die Parameter dabei Potenzgesetzen.

Complex chemical dynamics through engineering-like methods

Most of the problems in modern structural design can be described with a set of equation; solutions of these mathematical models can lead the engineer and designer to get info during the design stage. The same holds true for physical-chemistry; this branch of chemistry uses mathematics and physics in order to explain real chemical phenomena. In this work two extremely different chemical processes will be studied; the dynamic of an artificial molecular motor and the generation and propagation of the nervous signals between excitable cells and tissues like neurons and axons. These two processes, in spite of their chemical and physical differences, can be both described successfully by partial differential equations, that are, respectively the Fokker-Planck equation and the Hodgkin and Huxley model. With the aid of an advanced engineering software these two processes have been modeled and simulated in order to extract a lot of physical informations about them and to predict a lot of properties that can be, in future, extremely useful during the design stage of both molecular motors and devices which rely their actions on the nervous communications between active fibres.

Identification and classification of 16 new kinesin superfamily (KIF) proteins in mouse genome

KIF (kinesin superfamily) proteins are microtubule-dependent molecular motors that play important roles in intracellular transport and cell division. The extent to which KIFs are involved in various transporting phenomena, as well as their regulation mechanism, are unknown. The identification of 16 new KIFs in this report doubles the existing number of KIFs known in the mouse. Conserved nucleotide sequences in the motor domain were amplified by PCR using cDNAs of mouse nervous tissue, kidney, and small intestine as templates. The new KIFs were studied with respect to their expression patterns in different tissues, chromosomal location, and molecular evolution. Our results suggest that (i) there is no apparent tendency among related subclasses of KIFs of cosegregation in chromosomal mapping, and (ii) according to their tissue distribution patterns, KIFs can be divided into two classes–i.e., ubiquitous and specific tissue-dominant. Further characterization of KIFs may elucidate unknown fundamental phenomena underlying intracellular transport. Finally, we propose a straightforward nomenclature system for the members of the mouse kinesin superfamily.

A conventional myosin motor drives neurite outgrowth

Neuritic outgrowth is a striking example of directed motility, powered through the actions of molecular motors. Members of the myosin superfamily of actin-associated motors have been implicated in this complex process. Although conventional myosin II is known to be present in neurons, where it is localized at the leading edge of growth cones and in the cell cortex close to the plasma membrane, its functional involvement in growth cone motility has remained unproven. Here, we show that antisense oligodeoxyribonucleotides, complementary to a specific isoform of conventional myosin (myosin IIB), attenuate filopodial extension whereas sense and scrambled control oligodeoxyribonucleotides have no effect. Attenuation is shown to be reversible, neurite outgrowth being restored after cessation of the antisense regimen. Myosin IIB mRNA was present during active neurite extension, but levels were minimal in phenotypically rounded cells before neurite outgrowth and message levels decreased during antisense treatment. By contrast, the myosin IIA isoform is shown to be expressed constitutively both before and during neurite outgrowth and throughout exposure to myosin IIB antisense oligodeoxyribonucleotides. These results provide direct evidence that a conventional two-headed myosin is required for growth cone motility and is responsible, at least in part, for driving neuritic process outgrowth.

Homologous pairing in stretched supercoiled DNA

By using elastic measurements on single DNA molecules, we show that stretching a negatively supercoiled DNA activates homologous pairing in physiological conditions. These experiments indicate that a stretched unwound DNA locally denatures to alleviate the force-driven increase in torsional stress. This is detected by hybridization with 1 kb of homologous single-stranded DNA probes. The stretching force involved (≈2 pN) is small compared with those typically developed by molecular motors, suggesting that this process may be relevant to DNA processing in vivo. We used this technique to monitor the progressive denaturation of DNA as it is unwound and found that distinct, stable denaturation bubbles formed, beginning in A+T-rich regions.

Reciprocal electromechanical properties of rat prestin: The motor molecule from rat outer hair cells

Cochlear outer hair cells (OHCs) are responsible for the exquisite sensitivity, dynamic range, and frequency-resolving capacity of the mammalian hearing organ. These unique cells respond to an electrical stimulus with a cycle-by-cycle change in cell length that is mediated by molecular motors in the cells' basolateral membrane. Recent work identified prestin, a protein with similarity to pendrin-related anion transporters, as the OHC motor molecule. Here we show that heterologously expressed prestin from rat OHCs (rprestin) exhibits reciprocal electromechanical properties as known for the OHC motor protein. Upon electrical stimulation in the microchamber configuration, rprestin generates mechanical force with constant amplitude and phase up to a stimulus frequency of at least 20 kHz. Mechanical stimulation of rprestin in excised outside-out patches shifts the voltage dependence of the nonlinear capacitance characterizing the electrical properties of the molecule. The results indicate that rprestin is a molecular motor that displays reciprocal electromechanical properties over the entire frequency range relevant for mammalian hearing.

Simple mechanochemistry describes the dynamics of kinesin molecules

Recently, Block and coworkers [Visscher, K., Schnitzer, M. J., & Block, S. M. (1999) Nature (London) 400, 184–189 and Schnitzer, M. J., Visscher, K. & Block, S. M. (2000) Nat. Cell Biol. 2, 718–723] have reported extensive observations of individual kinesin molecules moving along microtubules in vitro under controlled loads, F = 1 to 8 pN, with [ATP] = 1 μM to 2 mM. Their measurements of velocity, V, randomness, r, stalling force, and mean run length, L, reveal a need for improved theoretical understanding. We show, presenting explicit formulae that provide a quantitative basis for comparing distinct molecular motors, that their data are satisfactorily described by simple, discrete-state, sequential stochastic models. The simplest (N = 2)-state model with fixed load-distribution factors and kinetic rate constants concordant with stopped-flow experiments, accounts for the global (V, F, L, [ATP]) interdependence and, further, matches relative acceleration observed under assisting loads. The randomness, r(F,[ATP]), is accounted for by a waiting-time distribution, ψ\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document} \begin{equation*}{\mathrm{_{1}^{+}}}\end{equation*}\end{document}(t), [for the transition(s) following ATP binding] with a width parameter ν ≡ 〈t〉2/〈(Δt)2〉≃2.5, indicative of a dispersive stroke of mechanicity ≃0.6 or of a few (≳ν − 1) further, kinetically coupled states: indeed, N = 4 (but not N = 3) models do well. The analysis reveals: (i) a substep of d0 = 1.8–2.1 nm on ATP binding (consistent with structurally based suggestions); (ii) comparable load dependence for ATP binding and unbinding; (iii) a strong load dependence for reverse hydrolysis and subsequent reverse rates; and (iv) a large (≳50-fold) increase in detachment rate, with a marked load dependence, following ATP binding.

Three lenses into the cardiac dyad: calcium signaling, microtubule trafficking, and junctional sarcoplasmic reticular mobility

Thesis (Ph.D.)--University of Washington, 2016-06

SNX4 coordinates endosomal sorting of TfnR with dynein-mediated transport into the endocytic recycling compartment

SNX-BAR proteins are a sub-family of sorting nexins implicated in endosomal sorting. Here, we establish that through its phox homology (PX) and Bin-Amphiphysin-Rvs (BAR) domains, sorting nexin-4 (SNX4) is associated with tubular and vesicular elements of a compartment that overlaps with peripheral early endosomes and the juxtanuclear endocytic recycling compartment (ERC). Suppression of SNX4 perturbs transport between these compartments and causes lysosomal degradation of the transferrin receptor (TfnR). Through an interaction with KIBRA, a protein previously shown to bind dynein light chain 1, we establish that SNX4 associates with the minus end-directed microtubule motor dynein. Although suppression of KIBRA and dynein perturbs early endosome-to-ERC transport, TfnR sorting is maintained. We propose that by driving membrane tubulation, SNX4 coordinates iterative, geometric-based sorting of the TfnR with the long-range transport of carriers from early endosomes to the ERC. Finally, these data suggest that by associating with molecular motors, SNX-BAR proteins may coordinate sorting with carrier transport between donor and recipient membranes.

Molecular sliding filament model for muscular contraction based on multiscale investigation

A multiscale approach that bridges the biophysics of the actin molecules at nanoscale and the biomechanics of actin filament at microscale level is developed and used to evaluate the mechanical performances of actin filament bundles. In order to investigate the contractile properties of skeletal muscle which is induced by the protein motor of myosin, a molecular model is proposed in the prediction of the dynamic behaviors of skeletal muscle based on classic sliding filament model. Randomly distributed myosin motors are applied on a 2.2 μm long sarcomere, whose principal components include actin and myosin filaments. It can be found that, the more myosin motors on the sarcomere, the faster the sarcomere contracts. The result demonstrates that the sarcomere shortening speed cannot increase infinitely by the modulation of myosin, thus providing insight into the self-protective properties of skeletal muscles. This molecular filament sliding model provides a theoretical way to evaluate the properties of skeletal muscles, and contributes to the understandings of the molecular mechanisms in the physiological phenomenon of muscular contraction.