Alvaro H. Crevenna

Lifeact: a versatile marker to visualize F-actin.

Live imaging of the actin cytoskeleton is crucial for the study of many fundamental biological processes, but current approaches to visualize actin have several limitations. Here we describe Lifeact, a 17-amino-acid peptide, which stained filamentous actin (F-actin) structures in eukaryotic cells and tissues. Lifeact did not interfere with actin dynamics in vitro and in vivo and in its chemically modified peptide form allowed visualization of actin dynamics in nontransfectable cells.

Processive Movement of MreB-Associated Cell Wall Biosynthetic Complexes in Bacteria

Bacteria elongation involves moving synthetic complexes around the cell wall. The peptidoglycan cell wall and the actin-like MreB cytoskeleton are major determinants of cell shape in rod-shaped bacteria. The prevailing model postulates that helical, membrane-associated MreB filaments organize elongation-specific peptidoglycan-synthesizing complexes along sidewalls. We used total internal reflection fluorescence microscopy to visualize the dynamic relation between MreB isoforms and cell wall synthesis in live Bacillus subtilis cells. During exponential growth, MreB proteins did not form helical structures. Instead, together with other morphogenetic factors, they assembled into discrete patches that moved processively along peripheral tracks perpendicular to the cell axis. Patch motility was largely powered by cell wall synthesis, and MreB polymers restricted diffusion of patch components in the membrane and oriented patch motion.

Cortical actin dynamics driven by formins and myosin V

Cell morphogenesis requires complex and rapid reorganization of the actin cytoskeleton. The budding yeast Saccharomyces cerevisiae is an invaluable model system for studying molecular mechanisms driving actin dynamics. Actin cables in yeast are formin-generated linear actin arrays that serve as tracks for directed intracellular transport by type V myosins. Cables are constantly reorganized throughout the cell cycle but the molecular basis for such dynamics remains poorly understood. By combining total internal reflection microscopy, quantitative image analyses and genetic manipulations we identify kinetically distinct subpopulations of cables that are differentially driven by formins and myosin. Bni1 drives elongation of randomly oriented actin cables in unpolarized cells, whereas both formins Bnr1 and Bni1 mediate slower polymerization of cables in polarized cells. Type V myosin Myo2 surprisingly acts as a motor for translational cable motility along the cell cortex. During polarization, cells change from fast to slow cable dynamics through spatio-temporal regulation of Bni1, Bnr1 and Myo2. In summary, we identify molecular mechanisms for the regulation of cable dynamics and suggest that fast actin reorganization is necessary for fidelity of cell polarization.

Influence of protein kinases on the osmosensitive release of taurine from cerebellar granule neurons.

The role of phosphorylation events on the activation and modulation of the osmosensitive (3)H-taurine release (OTR) was examined in cultured cerebellar granule neurons (CGN) stimulated with 30% hyposmotic solutions. OTR was not decreased when [Ca(2+)](i) rise evoked by hyposmolarity was prevented by EGTA-AM (50 microM) or depleted by treatment with 1 microM ionomycin in Ca(2+)-free medium. Accordingly, OTR was not inhibited by Ca(2+)-dependent signaling events. The calmodulin (CAM) blocker W-7 (50 microM) potentiated OTR while the Ca(2+)/CAM kinase blocker KN-93 (10 microM) was without effect. Blockade of PKC by H-7, H-8 (50 microM) and Gö6976 (1 microM), as well as activation by phorbol myristate acetate (PMA) (100 nM) did not influence OTR, but chronic treatment to down regulate PKC decreased it by 30%. Forskolin (20 microM) and 8-BrcAMP (10 microM) did not change OTR. Protein tyrosine phosphorylation seems to be of crucial importance in the activation and modulation of OTR, as it was markedly inhibited (90%) by tyrphostine A23 (50 microM) and potentiated by the tyrosine phosphatase inhibitor ortho-vanadate (100 microM). The PI3 kinase blocker wortmannin 100 nM essentially abolished OTR but LY294002 (10-100 microM) was without effect. This difference may be accounted for PI3K isoforms in neurons with different sensitivity to the blockers. Alternatively, the effect of wortmannin may be exerted not in PI3 kinase but instead on phospholipases, which are also sensitive to this blocker. The hyposmotic stimulus induced activation of Erk1/Erk2, but blockade of this effect by PD 98059 (50 microM) only marginally decreased OTR suggesting that the Erk1/Erk2 is an epiphenomenon, not directly involved in OTR activation.

Effects of Hofmeister Ions on the α-Helical Structure of Proteins

The molecular conformation of proteins is sensitive to the nature of the aqueous environment. In particular, the presence of ions can stabilize or destabilize (denature) protein secondary structure. The underlying mechanisms of these actions are still not fully understood. Here, we combine circular dichroism (CD), single-molecule Förster resonance energy transfer, and atomistic computer simulations to elucidate salt-specific effects on the structure of three peptides with large α-helical propensity. CD indicates a complex ion-specific destabilization of the α-helix that can be rationalized by using a single salt-free computer simulation in combination with the recently introduced scheme of ion-partitioning between nonpolar and polar peptide surfaces. Simulations including salt provide a molecular underpinning of this partitioning concept. Furthermore, our single-molecule Förster resonance energy transfer measurements reveal highly compressed peptide conformations in molar concentrations of NaClO(4) in contrast to strong swelling in the presence of GdmCl. The compacted states observed in the presence of NaClO(4) originate from a tight ion-backbone network that leads to a highly heterogeneous secondary structure distribution and an overall lower α-helical content that would be estimated from CD. Thus, NaClO(4) denatures by inducing a molten globule-like structure that seems completely off-pathway between a fully folded helix and a coil state.

Secondary Structure and Compliance of a Predicted Flexible Domain in Kinesin-1 Necessary for Cooperation of Motors

Although the mechanism by which a kinesin-1 molecule moves individually along a microtubule is quite well-understood, the way that many kinesin-1 motor proteins bound to the same cargo move together along a microtubule is not. We identified a 60-amino-acid-long domain, termed Hinge 1, in kinesin-1 from Drosophila melanogaster that is located between the coiled coils of the neck and stalk domains. Its deletion reduces microtubule gliding speed in multiple-motor assays but not single-motor assays. Hinge 1 thus facilitates the cooperation of motors by preventing them from impeding each other. We addressed the structural basis for this phenomenon. Video-microscopy of single microtubule-bound full-length motors reveals the sporadic occurrence of high-compliance states alternating with longer-lived, low-compliance states. The deletion of Hinge 1 abolishes transitions to the high-compliance state. Based on Fourier transform infrared, circular dichroism, and fluorescence spectroscopy of Hinge 1 peptides, we propose that low-compliance states correspond to an unexpected structured organization of the central Hinge 1 region, whereas high-compliance states correspond to the loss of that structure. We hypothesize that strain accumulated during multiple-kinesin motility populates the high-compliance state by unfolding helical secondary structure in the central Hinge 1 domain flanked by unordered regions, thereby preventing the motors from interfering with each other in multiple-motor situations.

Structural basis for the extended CAP-Gly domains of p150(glued) binding to microtubules and the implication for tubulin dynamics

Significance This study presents a direct visualization of the microtubule-p150glued(CAP-Gly) complex by cryo-EM and seeks to describe the molecular mechanism of the control of tubulin dynamics by p150 CAP-Gly. It highlights the neutralization of the acidic tubulin surface by the basic extensions of CAP-Gly, resulting in the activation of tubulin polymerization. In the condition where the lateral association is impeded (i.e., at low temperature), the extended CAP-Gly domain induces tubulin dimers to connect longitudinally. The two directional modes of self-association of tubulin suggest a foundation for its dynamic behavior at the tip of microtubules and its regulation. p150glued belongs to a group of proteins accumulating at microtubule plus ends (+TIPs). It plays a key role in initiating retrograde transport by recruiting and tethering endosomes and dynein to microtubules. p150glued contains an N-terminal microtubule-binding cytoskeleton-associated protein glycine-rich (CAP-Gly) domain that accelerates tubulin polymerization. Although this copolymerization is well-studied using light microscopic techniques, structural consequences of this interaction are elusive. Here, using electron-microscopic and spectroscopic approaches, we provide a detailed structural view of p150glued CAP-Gly binding to microtubules and tubulin. Cryo-EM 3D reconstructions of p150glued-CAP-Gly complexed with microtubules revealed the recognition of the microtubule surface, including tubulin C-terminal tails by CAP-Gly. These binding surfaces differ from other retrograde initiation proteins like EB1 or dynein, which could facilitate the simultaneous attachment of all accessory components. Furthermore, the CAP-Gly domain, with its basic extensions, facilitates lateral and longitudinal interactions of tubulin molecules by covering the tubulin acidic tails. This shielding effect of CAP-Gly and its basic extensions may provide a molecular basis of the roles of p150glued in microtubule dynamics.

Regulating Contractility of the Actomyosin Cytoskeleton by pH

The local interaction of F-actin with myosin-II motor filaments and crosslinking proteins is crucial for the force generation, dynamics, and reorganization of the intracellular cytoskeleton. By using a bottom-up approach, we are able to show that the contractility of reconstituted active actin systems is tightly controlled by the local pH. The pH-dependent intrinsic crossbridge strength of myosin-II is identified to account for a sharp transition of the actin/myosin-II activity from noncontractile to contractile by a change in pH of only 0.1. This pH-dependent contractility is a generic feature, which is observed in all studied crosslinked actin/myosin-II systems. The specific type and concentration of crosslinking protein allows one to sensitively adjust the range of pH where contraction occurs, which can recover the behavior found in Xenopus laevis oocyte extracts. Small variations in pH provide a mechanism of controlling the contractility of cytoskeletal structures, which can be expected to have broad implications in our understanding of cytoskeletal regulation.

Electrostatics control actin filament nucleation and elongation kinetics

Background: pH plays important roles in cellular morphogenesis, but how actin polymer dynamics are affected by pH is not well understood. Results: Actin filament nucleation and elongation are strongly enhanced at acidic pH due to a reduction of charge repulsion. Conclusion: Actin filament dynamics in vitro and in vivo are strongly influenced by electrostatics. Significance: Cellular pH homeostasis will impact directly on actin dynamics. The actin cytoskeleton is a central mediator of cellular morphogenesis, and rapid actin reorganization drives essential processes such as cell migration and cell division. Whereas several actin-binding proteins are known to be regulated by changes in intracellular pH, detailed information regarding the effect of pH on the actin dynamics itself is still lacking. Here, we combine bulk assays, total internal reflection fluorescence microscopy, fluorescence fluctuation spectroscopy techniques, and theory to comprehensively characterize the effect of pH on actin polymerization. We show that both nucleation and elongation are strongly enhanced at acidic pH, with a maximum close to the pI of actin. Monomer association rates are similarly affected by pH at both ends, although dissociation rates are differentially affected. This indicates that electrostatics control the diffusional encounter but not the dissociation rate, which is critical for the establishment of actin filament asymmetry. A generic model of protein-protein interaction, including electrostatics, explains the observed pH sensitivity as a consequence of charge repulsion. The observed pH effect on actin in vitro agrees with measurements of Listeria propulsion in pH-controlled cells. pH regulation should therefore be considered as a modulator of actin dynamics in a cellular environment.

Cofilin recruits F-actin to SPCA1 and promotes Ca2+-mediated secretory cargo sorting

The cofilin CFL-1 recruits actin to the P-type calcium ATPase SPCA1 at the trans-Golgi network, thereby activating the ATPase, promoting Ca2+ influx, and driving secretory cargo sorting.