Pretreatment with cholesterol-loaded cyclodextrins prevents loss of motility associated proteins during cryopreservation of addra gazelle (Nanger dama ruficollis) spermatozoa.

Sperm cryopreservation is challenging, often resulting in irreversible damage to spermatozoa, as indicated by decreased motility, viability, and/or acrosomal integrity. Developing cryopreservation protocols for gametes of endangered species compounds the complexity of technique optimization; samples are difficult to obtain and numbers are limited. Cryopreservation of sperm collected from the critically endangered addra gazelle (Nanger dama ruficollis), a member of the Bovidae family, resulted in significant loss of motility, which was prevented by pretreatment with cholesterol-loaded cyclodextrin (CLC). This study investigated the proteome of sperm (fresh and cryopreserved), processed in the absence and presence of 0.5 mg/ml CLC in the addra gazelle. The proteome of Bos taurus, the closest domestic relative, was used as a reference. Mass spectrometry analysis of the addra gazelle sperm proteome revealed 287 proteins. The concentrations of 85 proteins differed between fresh and frozen/thawed samples; nearly all were decreased. Most were associated with metabolic processes, specifically glycolysis, which may explain the decrease in post-thaw motility observed in this species. CLC pretreatment partially prevented the loss of various proteins involved in metabolism including CAPZB (gene = CAPZB), HS90A (gene = HSP90AA1), and PGAM2 (gene = PGAM2). To our knowledge, this is the first study to evaluate the proteome of any wild bovids' sperm, and the first to compare protein levels in sperm pretreated with CLC.

Interplay of active processes modulates tension and drives phase transition in self-renewing, motor-driven cytoskeletal networks.

The actin cytoskeleton--a complex, nonequilibrium network consisting of filaments, actin-crosslinking proteins (ACPs) and motors--confers cell structure and functionality, from migration to morphogenesis. While the core components are recognized, much less is understood about the behaviour of the integrated, disordered and internally active system with interdependent mechano-chemical component properties. Here we use a Brownian dynamics model that incorporates key and realistic features--specifically actin turnover, ACP (un)binding and motor walking--to reveal the nature and underlying regulatory mechanisms of overarching cytoskeletal states. We generate multi-dimensional maps that show the ratio in activity of these microscopic elements determines diverse global stress profiles and the induction of nonequilibrium morphological phase transition from homogeneous to aggregated networks. In particular, actin turnover dynamics plays a prominent role in tuning stress levels and stabilizing homogeneous morphologies in crosslinked, motor-driven networks. The consequence is versatile functionality, from dynamic steady-state prestress to large, pulsed constrictions.

Chronic alcohol exposure affects the cell components involved in membrane traffic in neuronal dendrites.

The specific traffic of the membrane components in neurons is a major requirement to establish and maintain neuronal domains-the axonal and the somatodendritic domains-and their polarized morphology. Unlike axons, dendrites contain membranous organelles, which are involved in the secretory pathway, including the endoplasmic reticulum, the Golgi apparatus and post-Golgi apparatus carriers, the cytoskeleton, and plasma membrane. A variety of molecules and factors are also involved in this process. Previous studies have shown that chronic alcohol exposure negatively affects several of these cell components, such as the Golgi apparatus or cytoskeleton in neurons. Yet very little information is available on the possible effects of this exposure on the remaining cell elements involved in intracellular trafficking in neurons, particularly in dendrites. By qualitative and quantitative electron microscopy, immunofluorescence and immunoblotting, we herein show that chronic exposure to moderate levels (30 mM) of ethanol in cultured neurons reduces the volume and surface density of the rough endoplasmic reticulum, and increases the levels of GRP78, a chaperone involved in endoplasmic reticulum stress. Ethanol also significantly diminishes the proportion of neurons that show an extension of Golgi into dendrites and dendritic Golgi outposts, a structure present exclusively in longer, thicker apical dendrites. Both Golgi apparatus types were also fragmented into a large number of cells. We also investigated the effect of alcohol on the levels of microtubule-based motor proteins KIF5, KIF17, KIFC2, dynein, and myosin IIb, responsible for transporting different cargoes in dendrites. Of these, alcohol differently affects several of them by lowering dynein and raising KIF5, KIFC2, and myosin IIb. These results, together with other previously published ones, suggest that practically all the protein trafficking steps in dendrites are altered to a greater or lesser extent by chronic alcohol exposure in neuronal cells, which may have negative repercussions for the development and maintenance of their polarized morphology and function.

Blood electrolytes exhibit a strong influence on the mobility of artificial catalytic microengines.

The developments in biomedical sciences foresee the inclusion of self-propelled catalytic micromotors for in vivo therapeutic strategies in the near future. We show here that blood electrolytes, such as Na(+), K(+), Ca(2+), Cl(-), SO4(2-) and phosphates, decrease the mobility of the Pt catalyzed tubular microjets. This effect is significant and in many cases, the microjets are completely disabled at physiologically relevant concentrations of the ions. A strategy to counterbalance this negative influence is suggested. These findings have a strong influence in the field of bubble-propelled artificial micromotors, where applications in blood are often envisioned.

The excluded volume effect induced by poly(ethylene glycol) modulates the motility of actin filaments interacting with myosin.

To examine the motility of actomyosin complexes in the presence of high concentrations of polymers, we investigated the effect of poly(ethylene glycol) on the sliding velocities of actin filaments and regulated thin filaments on myosin molecules in the presence of ATP. Increased concentrations and relative molecular masses of poly(ethylene glycol) decreased the sliding velocities of actin and regulated thin filaments. The decreased ratio of velocity in regulated thin filaments at - log[Ca(2+) ] of 4 was higher than that of actin filaments. Furthermore, in the absence of Ca(2+) , regulated thin filaments were moderately motile in the presence of poly(ethylene glycol). The excluded volume change (∆V), defined as the change in water volume surrounding actomyosin during the interactions, was estimated by determining the relationship between osmotic pressure exerted by poly(ethylene glycol) and the decreased ratio of the velocities in the presence and absence of poly(ethylene glycol). The ∆V increased up to 3.7 × 10(5) Å(3) as the Mr range of poly(ethylene glycol) was increased up to 20,000. Moreover, the ∆V for regulated thin filaments was approximately two-fold higher than that of actin filaments. This finding suggests that differences in the conformation of filaments according to whether troponin-tropomyosin complexes lie on actin filaments alter the ∆V during interactions of actomyosin complexes and influence motility.

Small peptide inhibitors disrupt a high-affinity interaction between cytoplasmic dynein and a viral cargo protein.

Several viruses target the microtubular motor system in early stages of the viral life cycle. African swine fever virus (ASFV) protein p54 hijacks the microtubule-dependent transport by interaction with a dynein light chain (DYNLL1/DLC8). This was shown to be a high-affinity interaction, and the residues gradually disappearing were mapped on DLC8 to define a putative p54 binding surface by nuclear magnetic resonance (NMR) spectroscopy. The potential of short peptides targeting the binding domain to disrupt this high-affinity protein-protein interaction was assayed, and a short peptide sequence was shown to bind and compete with viral protein binding to dynein. Given the complexity and number of proteins involved in cellular transport, the prevention of this viral-DLC8 interaction might not be relevant for successful viral infection. Thus, we tested the capacity of these peptides to interfere with viral infection by disrupting dynein interaction with viral p54. Using this approach, we report on short peptides that inhibit viral growth.

Microinjected F-actin into dividing newt eggs moves toward the next cleavage furrow together with Ca2+ stores with inositol 1,4,5-trisphosphate receptor in a microtubule- and microtubule motor-dependent manner.

It has been reported that F-actin is transported to the presumptive cleavage furrow along the cortex during anaphase-cytokinesis, an event termed cortical actin flow in animal cultured cells. The motor source has remained unknown. We reported that Ca2+ stores with IP3 receptor (IP3R) was re-distributed from the polar cortex during metaphase to the presumptive cleavage furrow just before the onset of furrowing, and that Ca2+ stores with IP3R microinjected into dividing newt eggs moved toward the presumptive cleavage furrow during anaphase-cytokinesis in a microtubule-dependent manner, and that Ca2+ store-enriched microsome fractions induced the cleavage furrow as the putative cleavage stimulus. Because the distribution of F-actin and Ca2+ stores with IP3R during metaphase to cytokinesis is similar, we considered that this cortical actin flow may be powered by transportation of Ca2+ stores with IP3R. Purified F-actin labeled with phalloidin-rhodamine was microinjected into the dividing newt eggs and the eggs observed under a confocal microscope. We found that the microinjected F-actin moved linearly toward the next cleavage furrow and that this movement was blocked by nocodazole, microtubule-depolarizing agent and AMP-PNP, a blocking agent of microtubule motors. Co-microinjected rhodamine-labeled F-actin and sacro/endoplasmic reticulum Ca2+-ATPase (SERCA)-GFP-labeled Ca2+ stores with IP3R co-moved and co-accumulated to the next cleavage furrow. These results strongly suggest that Ca2+ stores with IP3R, which is transferred by microtubule-based motility as cleavage stimulus, act as an F-actin translocator.

Peroxynitrite inhibits myofibrillar protein function in an in vitro assay of motility.

We determined the effects of peroxynitrite (ONOO-) on cardiac myosin, actin, and thin filaments in order to more clearly understand the impact of this reactive compound in ischemia/reperfusion injury and heart failure. Actin filaments, native thin filaments, and alpha-cardiac myosin from rat hearts were exposed to ONOO- in the presence of 2 mM bicarbonate. Filament velocities over myosin, calcium sensitivity, and relative force generated by myosin were assessed in an in vitro motility assay in the absence of reducing agents. ONOO- concentrations > or =10 microM significantly reduced the velocities of thin filaments or bare actin filaments over alpha-cardiac myosin when any of these proteins were exposed individually. These functional deficits were linearly related to the degree of tyrosine nitration, with myosin being the most sensitive. However, at 10 microM ONOO- the calcium sensitivity of thin filaments remained unchanged. Cotreatment of myosin and thin filaments, analogous to the in vivo situation, resulted in a significantly greater functional deficit. The load supported by myosin after ONOO- exposure was estimated using mixtures experiments to be increased threefold. These data suggest that nitration of myofibrillar proteins can contribute to cardiac contractile dysfunction in pathologic states in which ONOO- is liberated.

Nordihydroguaiaretic acid affects multiple dynein-dynactin functions in interphase and mitotic cells.

Nordihydroguaiaretic acid (NDGA), a well known lipoxygenase inhibitor, actually has pleiotropic effects on cells, which include cell proliferation, apoptosis, differentiation, and chemotaxis. We and others have shown previously that this compound causes Golgi disassembly by an unknown mechanism. In this study, we show that, in parallel with Golgi disassembly, NDGA induces the accumulation of the microtubule minus-end-directed motor dynein-dynactin complex at the centrosome, where microtubules minus-ends lie. Concomitant with this accumulation, dynein-dynactin-interacting proteins, such as ZW10 and EB1, were also redistributed to the centrosomal region. In cells where microtubules were depolymerized by nocodazole, NDGA promoted the formation of filaments consisting of dynein-dynactin and its interacting proteins, suggesting that it stimulates the association of these proteins in an ordered, not random, manner. Loss of dynactin function abolished not only NDGA-induced redistribution in intact cells but also filament formation in nocodazole-treated cells. The latter finding implies that dynactin is a key molecule for the association between dynein-dynactin and its interacting proteins. In mitotic cells, NDGA induced robust accumulation of dyneindynactin and its interacting proteins at the spindle poles. These results taken together suggest that NDGA perturbs membrane traffic by affecting the function of the microtubule motor dynein-dynactin complex and its auxiliary proteins. To our knowledge, NDGA is the first case of a reagent that can modulate dynein-dynactin-related processes.

A microrotary motor powered by bacteria.

Biological molecular motors have a number of unique advantages over artificial motors, including efficient conversion of chemical energy into mechanical work and the potential for self-assembly into larger structures, as is seen in muscle sarcomeres and bacterial and eukaryotic flagella. The development of an appropriate interface between such biological materials and synthetic devices should enable us to realize useful hybrid micromachines. Here we describe a microrotary motor composed of a 20-mum-diameter silicon dioxide rotor driven on a silicon track by the gliding bacterium Mycoplasma mobile. This motor is fueled by glucose and inherits some of the properties normally attributed to living systems.

Arginine inhibits Na-driven flagellar motors of alkaliphilic Bacillus.

L-arginine has attracted a great deal of attention as an agent for refolding denatured proteins, and the mildness of its effects offer hope for a wide range of potential applications for this substance, including medicines with few side effects. We report that both L- and D-arginine inhibits Na+-driven flagellar motors of alkaliphilic Bacillus by competing with Na+, which we take as evidence that arginine specifically binds to a molecular target.

Effect of spermine and DNase on DNA release from bacteriophage T5.

Bacterial viruses (bacteriophages) consist of nucleic acid protected by a protein envelope called capsid. At the start of infection, the phage genome is translocated into the bacterial cytoplasm. In vitro (and also in vivo), this DNA release can be triggered by binding a specific receptor protein to the phage tail. The force responsible for the release arises from energy stored in the capsid due to strong confinement of the DNA. We show that this force can be modified by adding molecules like spermine that affect DNA conformation. The tetravalent cation spermine can reduce the pressure inside the capsid and induce condensation of the released DNA. We examine the effect of spermine on DNA ejection from phage T5 by using light scattering and gel electrophoresis to measure the amount of DNA remaining in the capsid at the end of ejection. We discuss the results in terms of free energy minimization and we demonstrate that the presence of a DNA condensate outside the phage generates an additional force pulling passively on the DNA remaining inside the capsid.

Ethanol perturbs the secretory pathway in astrocytes.

Ethanol exposure induces retention of glycoproteins in growing astrocytes. We examined the intracellular sites at which this retention occurs and investigated whether this effect is accompanied by alterations in the Golgi complex and microtubular system. We studied the effects of ethanol on the Golgi complex structure, as well as on the secretory pathway functionality by monitoring both the transport of the VSV-G protein and the protein levels of several molecules involved in the regulation of this pathway. Ethanol was found to delay VSV-G transport, modify Golgi complex morphology, and reduce the number of secretory vesicles. Moreover, ethanol affected the levels of mannosidase II, p58, betaCOP, rbet1, and several Rab GTPases. It also affected microtubule organization and polymerization and the levels of the motor proteins kinesin and dynein. Most of these effects were dose-dependent. These alterations, together with those previously reported concerning biosynthesis of glycoconjugates, provide novel insights into how ethanol impairs brain development.

Kinesin is involved in protecting nascent microtubules from disassembly after recovery from nocodazole treatment.

Upon recovery from nocodazole treatment, microtubules from cultured epithelial cells exhibit unusual properties: they re-grow as fast as any highly dynamic microtubule, but they are also protected against disassembly when challenged with nocodazole like the stable microtubules of steady-state cells. Exploring the mechanism that underlies this protection, we found that it was sensitive to ATP treatment and that it involved conventional kinesin. Kinesin localized at the growing end or along nascent microtubules. Its inhibition using a dominant-negative construct for cargo binding, or by micro-injecting an anti-kinesin heavy chain antibody that impairs motor activity, resulted in the partial or total loss of microtubule protection. Finally, in an ex vivo elongation assay, we found that kinesin also participates in the control of microtubule re-growth. Altogether, our findings suggest that kinesin is involved in an early microtubule protection process that is linked to the control of their dynamics during their early growth phase.

On membrane motor activity and chloride flux in the outer hair cell: lessons learned from the environmental toxin tributyltin.

The outer hair cell (OHC) underlies mammalian cochlea amplification, and its lateral membrane motor, prestin, which drives the cell's mechanical activity, is modulated by intracellular chloride ions. We have previously described a native nonselective conductance (G(metL)) that influences OHC motor activity via Cl flux across the lateral membrane. Here we further investigate this conductance and use the environmental toxin tributyltin (TBT) to better understand Cl-prestin interactions. Capitalizing on measures of prestin-derived nonlinear capacitance to gauge Cl flux across the lateral membrane, we show that the Cl ionophore TBT, which affects neither the motor nor G(metL) directly, is capable of augmenting the native flux of Cl in OHCs. These observations were confirmed using the chloride-sensitive dye MQAE. Furthermore, the compound's potent ability, at nanomolar concentrations, to equilibrate intra- and extracellular Cl concentrations is shown to surpass the effectiveness of G(metL) in promoting Cl flux, and secure a quantitative analysis of Cl-prestin interactions in intact OHCs. Using malate as an anion replacement, we quantify chloride effects on the nonlinear charge density and operating voltage range of prestin. Our data additionally suggest that ototoxic effects of organotins can derive from their disruption of OHC Cl homeostasis, ultimately interfering with anionic modulation of the mammalian cochlear amplifier. Notably, this observation identifies a new environmental threat for marine mammals by TBT, which is known to accumulate in the food chain.

Dynamic rearrangements of transvacuolar strands in BY-2 cells imply a role of myosin in remodeling the plant actin cytoskeleton.

Plant cells typically contain a large central vacuole that confines the cytoplasm and organelles to the periphery of the cell and the vicinity of the nucleus. These two domains are often connected by transvacuolar strands (TVS), thin tubular structures that traverse the vacuole. The TVS are thought to act as important transport routes for the distribution of organelles and metabolites, and also to play a role in the positioning of the nucleus. Most TVS depend on internal actin filaments for their existence, and rearrangements of TVS can therefore indicate modifications in the actin cytoskeleton. In this study we describe time-lapse observations of tobacco BY-2 suspension-cultured cells that document the dynamic behavior of TVS. The TVS formed, branched, and collapsed, and their attachment points in the nuclear or cortical cytoplasm, as well as on other TVS, moved around. These dynamic rearrangements were inhibited within 5 min by the myosin inhibitor 2,3-butanedione monoxime (BDM). In particular, the movements of TVS attachment points and the variations in TVS length were significantly reduced in the presence of the drug. Similarly, movements of the nucleus were reduced by two thirds in BDM-treated cells. The number of TVS, together with the number of attachment and branch points, also dropped during BDM treatment. All effects of BDM on TVS dynamics were reversible upon removal of the drug. These results suggest a role for myosin motors in the rearrangement of TVS, which is likely to occur through their interaction with actin filaments.

Mobile actin clusters and traveling waves in cells recovering from actin depolymerization.

At the leading edge of a motile cell, actin polymerizes in close apposition to the plasma membrane. Here we ask how the machinery for force generation at a leading edge is established de novo after the global depolymerization of actin. The depolymerization is accomplished by latrunculin A, and the reorganization of actin upon removal of the drug is visualized in Dictyostelium cells by total internal reflection fluorescence microscopy. The actin filament system is reorganized in three steps. First, F-actin assembles into globular complexes that move along the bottom surface of the cells at velocities up to 10 microm/min. These clusters are transient structures that eventually disassemble, fuse, or divide. In a second step, clusters merge into a contiguous zone at the cell border that spreads and gives rise to actin waves traveling on a planar membrane. Finally, normal cell shape and motility are resumed. These data show that the initiation of actin polymerization is separated in Dictyostelium from front protrusion, and that the coupling of polymerization to protrusion is a later step in the reconstitution of a leading edge.

Putting a brake on an autonomous DNA nanomotor.

A strategy was developed to reversibly switch on/off an autonomous DNA nanomotor that contains a DNA enzyme. The multiple RNA cleavage of the DNAzyme powered the motor to move, and a strand displacement mechanism provided the basis for a reversible brake to the motor.

Genotoxicity of inorganic mercury salts based on disturbed microtubule function.

This study investigated the hypothesis that the chromosomal genotoxicity of inorganic mercury results from interaction(s) with cytoskeletal proteins. Effects of Hg2+ salts on functional activities of tubulin and kinesin were investigated by determining tubulin assembly and kinesin-driven motility in cell-free systems. Hg2+ inhibits microtubule assembly at concentrations above 1 microM, and inhibition is complete at about 10 microM. In this range, the tubulin assembly is fully (up to 6 microM) or partially (~6-10 microM) reversible. The inhibition of tubulin assembly by mercury is independent of the anion, chloride or nitrate. The no-observed-effect-concentration for inhibition of microtubule assembly in vitro was 1 microM Hg2+, the IC50 5.8 microM. Mercury(II) salts at the IC50 concentrations partly inhibiting tubulin assembly did not cause the formation of aberrant microtubule structures. Effects of mercury salts on the functionality of the microtubule motility apparatus were studied with the motor protein kinesin. By using a "gliding assay" mimicking intracellular movement and transport processes in vitro, HgCl2 affected the gliding velocity of paclitaxel-stabilised microtubules in a clear dose-dependent manner. An apparent effect is detected at a concentration of 0.1 microM and a complete inhibition is reached at 1 microM. Cytotoxicity of mercury chloride was studied in V79 cells using neutral red uptake, showing an influence above 17 microM HgCl2. Between 15 and 20 microM HgCl2 there was a steep increase in cell toxicity. Both mercury chloride and mercury nitrate induced micronuclei concentration-dependently, starting at concentrations above 0.01 microM. CREST analyses on micronuclei formation in V79 cells demonstrated both clastogenic (CREST-negative) and aneugenic effects of Hg2+, with some preponderance of aneugenicity. A morphological effect of high Hg2+ concentrations (100 microM HgCl2) on the microtubule cytoskeleton was verified in V79 cells by immuno-fluorescence staining. The overall data are consistent with the concept that the chromosomal genotoxicity could be due to interaction of Hg2+ with the motor protein kinesin mediating cellular transport processes. Interactions of Hg2+ with the tubulin shown by in vitro investigations could also partly influence intracellular microtubule functions leading, together with the effects on the kinesin, to an impaired chromosome distribution as shown by the micronucleus test.

Fibroblasts regulate contractile force independent of MMP activity in 3D-collagen.

The extracellular matrix not only provides a structural scaffold for cells to inhabit but also forms a conduit by which mechanical information may be transmitted. Fibroblasts undergo a variety of changes when activated, including upregulating matrix metalloproteinase (MMP) activity and establishing a smooth muscle-like contractile apparatus. The relationship between MMP activity and matrix contraction has yet to be established. Here we report that inhibition of MMP activity correlates with a significant reduction in collagen gel contraction, however, force development does not change respective to MMP activity. These results suggest cellular controls of contractile forces are independent of MMP activity. Our results also raise the possibility that the material properties of the matrix dynamically change during remodeling.