Tim Scholz

Plus- and Minus-End Directed Microtubule Motors Bind Simultaneously to Herpes Simplex Virus Capsids Using Different Inner Tegument Structures

Many viruses depend on host microtubule motors to reach their destined intracellular location. Viral particles of neurotropic alphaherpesviruses such as herpes simplex virus 1 (HSV1) show bidirectional transport towards the cell center as well as the periphery, indicating that they utilize microtubule motors of opposing directionality. To understand the mechanisms of specific motor recruitment, it is necessary to characterize the molecular composition of such motile viral structures. We have generated HSV1 capsids with different surface features without impairing their overall architecture, and show that in a mammalian cell-free system the microtubule motors dynein and kinesin-1 and the dynein cofactor dynactin could interact directly with capsids independent of other host factors. The capsid composition and surface was analyzed with respect to 23 structural proteins that are potentially exposed to the cytosol during virus assembly or cell entry. Many of these proteins belong to the tegument, the hallmark of all herpesviruses located between the capsid and the viral envelope. Using immunoblots, quantitative mass spectrometry and quantitative immunoelectron microscopy, we show that capsids exposing inner tegument proteins such as pUS3, pUL36, pUL37, ICP0, pUL14, pUL16, and pUL21 recruited dynein, dynactin, kinesin-1 and kinesin-2. In contrast, neither untegumented capsids exposing VP5, VP26, pUL17 and pUL25 nor capsids covered by outer tegument proteins such as vhs, pUL11, ICP4, ICP34.5, VP11/12, VP13/14, VP16, VP22 or pUS11 bound microtubule motors. Our data suggest that HSV1 uses different structural features of the inner tegument to recruit dynein or kinesin-1. Individual capsids simultaneously accommodated motors of opposing directionality as well as several copies of the same motor. Thus, these associated motors either engage in a tug-of-war or their activities are coordinately regulated to achieve net transport either to the nucleus during cell entry or to cytoplasmic membranes for envelopment during assembly.

Mutation of the myosin converter domain alters cross-bridge elasticity.

Elastic distortion of a structural element of the actomyosin complex is fundamental to the ability of myosin to generate motile forces. An elastic element allows strain to develop within the actomyosin complex (cross-bridge) before movement. Relief of this strain then drives filament sliding, or more generally, movement of a cargo. Even with the known crystal structure of the myosin head, however, the structural element of the actomyosin complex in which elastic distortion occurs remained unclear. To assign functional relevance to various structural elements of the myosin head, e.g., to identify the elastic element within the cross-bridge, we studied mechanical properties of muscle fibers from patients with familial hypertrophic cardiomyopathy with point mutations in the head domain of the β-myosin heavy chain. We found that the Arg-719 → Trp (Arg719Trp) mutation, which is located in the converter domain of the myosin head fragment, causes an increase in force generation and fiber stiffness under isometric conditions by 48–59%. Under rigor and relaxing conditions, fiber stiffness was 45–47% higher than in control fibers. Yet, kinetics of active cross-bridge cycling were unchanged. These findings, especially the increase in fiber stiffness under rigor conditions, indicate that cross-bridges with the Arg719Trp mutation are more resistant to elastic distortion. The data presented here strongly suggest that the converter domain that forms the junction between the catalytic and the light-chain-binding domain of the myosin head is not only essential for elastic distortion of the cross-bridge, but that the main elastic distortion may even occur within the converter domain itself.

Tau Protein Diffuses along the Microtubule Lattice

Background: Tau protein is believed to be stationary while bound to microtubules. Results: Tau molecules can diffuse along microtubules over distances up to several micrometers. Conclusion: Tau diffusion on microtubules is a novel mechanism for Tau dispersion in cells. Significance: Modulation of Tau binding and diffusion on microtubules by local modifications of microtubules can provide a tool to target Tau to specific cellular compartments. Current models for the intracellular transport of Tau protein suggest motor protein-dependent co-transport with microtubule fragments and diffusion of Tau in the cytoplasm, whereas Tau is believed to be stationary while bound to microtubules and in equilibrium with free diffusion in the cytosol. Observations that members of the microtubule-dependent kinesin family show Brownian motion along microtubules led us to hypothesize that diffusion along microtubules could also be relevant in the case of Tau. We used single-molecule total internal reflection fluorescence microscopy to probe for diffusion of individual fluorescently labeled Tau molecules along microtubules. This allowed us to avoid the problem that microtubule-dependent diffusion could be masked by excess of labeled Tau in solution that might occur in in vivo overexpression experiments. We found that approximately half of the individually detected Tau molecules moved bidirectionally along microtubules over distances up to several micrometers. Diffusion parameters such as diffusion coefficient, interaction time, and scanned microtubule length did not change with Tau concentration. Tau binding and diffusion along the microtubule lattice, however, were sensitive to ionic strength and pH and drastically reduced upon enzymatic removal of the negatively charged C termini of tubulin. We propose one-dimensional Tau diffusion guided by the microtubule lattice as one possible additional mechanism for Tau distribution. By such one-dimensional microtubule lattice diffusion, Tau could be guided to both microtubule ends, i.e. the sites where Tau is needed during microtubule polymerization, independently of directed motor-dependent transport. This could be important in conditions where active transport along microtubules might be compromised.

Transport and diffusion of Tau protein in neurons

In highly polarized and elongated cells such as neurons, Tau protein must enter and move down the axon to fulfill its biological task of stabilizing axonal microtubules. Therefore, cellular systems for distributing Tau molecules are needed. This review discusses different mechanisms that have been proposed to contribute to the dispersion of Tau molecules in neurons. They include (1) directed transport along microtubules as cargo of tubulin complexes and/or motor proteins, (2) diffusion, either through the cytosolic space or along microtubules, and (3) mRNA-based mechanisms such as transport of Tau mRNA into axons and local translation. Diffusion along the microtubule lattice or through the cytosol appear to be the major mechanisms for axonal distribution of Tau protein in the short-to-intermediate range over distances of up to a millimetre. The high diffusion coefficients ensure that Tau can distribute evenly throughout the axonal volume as well as along microtubules. Motor protein-dependent transport of Tau dominates over longer distances and time scales. At low near-physiological levels, Tau is co-transported along with short microtubules from cell bodies into axons by cytoplasmic dynein and kinesin family members at rates of slow axonal transport.

Myosin-1C associates with microtubules and stabilizes the mitotic spindle during cell division

The mitotic spindle in eukaryotic cells is composed of a bipolar array of microtubules (MTs) and associated proteins that are required during mitosis for the correct partitioning of the two sets of chromosomes to the daughter cells. In addition to the well-established functions of MT-associated proteins (MAPs) and MT-based motors in cell division, there is increasing evidence that the F-actin-based myosin motors are important mediators of F-actin–MT interactions during mitosis. Here, we report the functional characterization of the long-tailed class-1 myosin myosin-1C from Dictyostelium discoideum during mitosis. Our data reveal that myosin-1C binds to MTs and has a role in maintenance of spindle stability for accurate chromosome separation. Both myosin-1C motor function and tail-domain-mediated MT–F-actin interactions are required for the cell-cycle-dependent relocalization of the protein from the cell periphery to the spindle. We show that the association of myosin-1C with MTs is mediated through the tail domain. The myosin-1C tail can inhibit kinesin motor activity, increase the stability of MTs, and form crosslinks between MTs and F-actin. These data illustrate that myosin-1C is involved in the regulation of MT function during mitosis in D. discoideum.

Inorganic Phosphate Binds to the Empty Nucleotide Binding Pocket of Conventional Myosin II

In muscle inorganic phosphate strongly decreases force generation in the presence of millimolar MgATP, whereas phosphate slows shortening velocity only at micromolar MgATP concentrations. It is still controversial whether reduction in shortening velocity by phosphate results from phosphate binding to the nucleotide-free myosin head or from binding of phosphate to an actomyosin-ADP state as postulated for the inhibition of force generation by phosphate. Because most single-molecule studies are performed at micromolar concentrations of MgATP where phosphate effects on movement are rather prominent, clarification of the mechanisms of phosphate inhibition is essential for interpretation of data in which phosphate is used in single molecule studies to probe molecular events of force generation and movement. In in vitro assays we found that inhibition of filament gliding by inorganic phosphate was associated with increased fragmentation of actin filaments. In addition, phosphate did not extend dwell times of Cy3-EDA-ATP (2′(3′)-O-[[2-[[6-[2-[3-(1-ethyl-1,3-dihydro-3,3-dimethyl-5-sulfo-2H-indol-2-ylidene)-1-propenyl]-3,3-dimethyl-5-sulfo-3H-indolio]-1-oxohexyl]amino]ethyl]carbamoyl]ATP) but reduced the number of Cy3-signals per field of view, approaching 50% at phosphate concentrations of 1–2 mm. Apparently, inhibition of movement does not result from binding of phosphate to an actomyosin-ADP intermediate as proposed by Hooft and coworkers (Hooft, A. M., Maki, E. J., Cox, K. K., and Baker, J. E. (2007) Biochemistry 46, 3513–3520) but, rather, from forming a strong-binding actomyosin-phosphate intermediate.

Processive behaviour of kinesin observed using micro-fabricated cantilevers.

The mechanical characterization of biomolecular motors requires force sensors with sub-piconewton resolution. The coupling of a nanoscale motor to this type of microscale sensors introduces structural deformations in the motor according to the thermally activated degrees of freedom of the sensor. At present, no simple solution is available to reduce these effects. Here, we exploit the advantages of micro-fabricated cantilevers to produce a force sensor with essentially one degree of freedom and a spring constant of 0.03 pN nm(-1) for the study of the molecular motor protein kinesin-1. During processive runs, the cantilever constrains the movement of the cargo binding domain of kinesin in a straight line, parallel to the microtubule track, and excludes specific reaction coordinates such as cargo rotation. In these conditions, we measured a step size of 8.0 ± 0.4 nm and a maximal unloaded velocity of 820 ± 80 nm s(-1) at saturated adenosine triphosphate (ATP) concentration. We concluded that the motor does not need to rotate its tail as it moves through consecutive stepping cycles.

Actin sliding on reconstituted myosin filaments containing only one myosin heavy chain isoform

We developed a technique to reconstitute myosin filaments containing only one myosin heavy chain (MyHC) isoform. Myosin was extracted from single skinned fibers of rabbit psoas muscle to ensure formation of filaments from only one MyHC isoform. Myosin filaments of up to about 20 μm in length were reconstituted by dialysing the extracted myosin against a buffer of slowly decreasing ionic strength. Length and diameter of the reconstituted myosin filaments were determined by electron microscopy. The reconstituted filaments were very heterogeneous in length, filament diameter was found to increase with length. The reconstituted myosin filaments were found to be functionaly bipolar like native thick filaments. Actin sliding towards the center of a reconstituted myosin filament occurred at 6.2 μm/s. Away from the center of these myosin filaments, i.e., in the unphysiological direction, actin-sliding velocity was found to be only 1.5 μm/s. We used these reconstituted myosin filaments to test whether ordered orientation and a more physiological environment for myosin molecules within reconstituted filaments can explain our previous finding that sliding velocity of actin filaments in in vitro motility assays with randomly attached myosin molecules extracted from single fibers is 4–8-fold slower than unloaded shortening velocity in muscle fibers even when experimental conditions and MyHC isoforms are identical (Thedinga E et al., (1999) J Muscle Res Cell Motil 20(8): 785–796).

Mechanical and kinetic properties of β-cardiac/slow skeletal muscle myosin

We aimed to establish reference parameters to identify functional effects of familial hypertrophic cardiomyopathy-related point mutations in the β-cardiac/slow skeletal muscle myosin heavy chain (β-cardiac/MyHC-1). We determined mechanical and kinetic parameters of the β-cardiac/MyHC-1 using human soleus muscle fibers that express the same myosin heavy chain (MyHC-1) as ventricular myocardium (β-cardiac). The observed parameters are compared to previously reported data for rabbit psoas muscle fibers. We found all of the examined kinetic parameters to be slower in soleus fibers than in rabbit psoas muscle. Somewhat surprisingly, however, we also found that the stiffness of the β-cardiac/MyHC-1 head domain is more than 3-fold lower than the stiffness of the fast isoform of psoas fibers. Furthermore, and different from rabbit psoas muscle, in human soleus fibers both the occupancy of force-generating cross-bridge states as well as the elastic extension of force-generating heads increase with temperature. Thus, a myosin head in the force generating states makes an increasing contribution to force with temperature. We support some of our fiber data by data from in vitro motility and optical trapping assays. Initial findings with FHC-related point mutations in the converter imply that the differences in stiffness of the head domain between the slow and fast isoform may well be due to particular differences in the amino acid sequence of the converter. We show that the slower kinetics may be linked to a larger flexibility of the β-cardiac/MyHC-1 isoform compared to fast MyHC isoforms.

The natural diterpene tonantzitlolone A and its synthetic enantiomer inhibit cell proliferation and kinesin-5 function.

Tonantzitlolone A, a diterpene isolated from the Mexican plant Stillingia sanguinolenta, shows cytostatic activity. Both the natural product tonantzitlolone A and its synthetic enantiomer induce monoastral spindle formation in cell experiments which indicates inhibitory activity on kinesin-5 mitotic motor molecules. These inhibitory effects on kinesin-5 could be verified in in vitro single-molecule motility assays, where both tonantzitlolones interfered with kinesin-5 binding to its cellular interaction partner microtubules in a concentration-dependent manner, yet with a larger effect of the synthetic enantiomer. In contrast to kinesin-5 inhibition, both tonantzitlolone A enantiomers did not affect conventional kinesin-1 function; hence tonantzitlolones are not unspecific kinesin inhibitors. The observed stronger inhibitory effect of the synthetic enantiomer demonstrates the possibility to enhance the overall moderate anti-proliferative effect of the lead compound tonantzitlolon A by chemical modification.