John Peloquin

Formation of filopodia-like bundles in vitro from a dendritic network.

We report the development and characterization of an in vitro system for the formation of filopodia-like bundles. Beads coated with actin-related protein 2/3 (Arp2/3)–activating proteins can induce two distinct types of actin organization in cytoplasmic extracts: (1) comet tails or clouds displaying a dendritic array of actin filaments and (2) stars with filament bundles radiating from the bead. Actin filaments in these bundles, like those in filopodia, are long, unbranched, aligned, uniformly polar, and grow at the barbed end. Like filopodia, star bundles are enriched in fascin and lack Arp2/3 complex and capping protein. Transition from dendritic to bundled organization was induced by depletion of capping protein, and add-back of this protein restored the dendritic mode. Depletion experiments demonstrated that star formation is dependent on Arp2/3 complex. This poses the paradox of how Arp2/3 complex can be involved in the formation of both branched (lamellipodia-like) and unbranched (filopodia-like) actin structures. Using purified proteins, we showed that a small number of components are sufficient for the assembly of filopodia-like bundles: Wiskott-Aldrich syndrome protein (WASP)–coated beads, actin, Arp2/3 complex, and fascin. We propose a model for filopodial formation in which actin filaments of a preexisting dendritic network are elongated by inhibition of capping and subsequently cross-linked into bundles by fascin.

Conjugation of fluorophores to tubulin

The use of fluorescently labeled proteins as probes of the structure and function of living cells began with the study of the components of the cytoskeleton1–4 and has become a powerful approach for studying the distribution and dynamics of these components5–7. Primary components of the cytoskeleton such as tubulin, actin and myosin are abundant and can be purified relatively easily in hundreds of milligram quantities from animal tissues8–11. Fluorophores containing amine or thiol reactive groups are used to selectively modify lysine or cysteine residues, respectively. It is essential that derivatization of the protein not result in loss of function. Methods for selecting active protein and removing unconjugated fluorophore are usually used after derivatization. The choice of fluorophore at any given wavelength of fluorescence emission should optimize fluorescence intensity and photostability. Tubulin is labeled in polymeric form to protect residues that are important for assembly and can be successfully conjugated with fluorescent derivatives of amine-reactive succinimidyl esters. Functional tubulin is selected by cycles of polymerization and depolymerization. By modifying the ratio of reactants, the Cy3-labeling protocol can also be used to derivatize tubulin with Cy3.5, Cy5, tetramethylrhodamine, X-rhodamine, Oregon Green 488, and Alexa 488, 568 and 594 succinimidyl esters. Although fluorescent fusion proteins have become widely used in imaging studies, fluorophore-labeled proteins remain the most versatile and direct tools for studying the dynamics and function of the cytoskeleton. This protocol describes the fluorescent conjugation of tubulin isolated from brain tissue.

Phosphorylation controls autoinhibition of cytoplasmic linker protein-170

CLIP-170 conformational changes are regulated by phosphorylation on S309 and S311 residues resulting in diminished binding of CLIP-170 for growing MT ends and p150Glued.

In Vitro Assembly of Filopodia‐Like Bundles

A breakthrough in understanding the mechanism of lamellipodial protrusion came from development of an in vitro model system, namely the rocketing movement of microbes and activated beads driven by actin comet tails (Cameron et al., 1999, 2000; Loisel et al., 1999; Theriot et al., 1994). As a model for investigation of the other major protrusive organelle, the filopodium, we developed in vitro systems for producing filopodia-like bundles (Vignjevic et al., 2003), one of which uses cytoplasmic extracts and another that reconstitutes like-like bundles from purified proteins. Beads coated with Arp2/3-activating proteins can induce two distinct types of actin organization in cytoplasmic extracts: (1) comet tails or clouds displaying a dendritic array of actin filaments and (2) stars with filament bundles radiating from the bead. Actin filaments in star bundles, like those in filopodia, are long, unbranched, aligned, uniformly polar, and grow at the barbed end. Like filopodia, star bundles are enriched in fascin and lack Arp2/3 complex and capping protein. Similar to cells, the transition from a dendritic (lamellipodial) to a bundled (filopodial) organization is induced by depletion of capping protein, and add-back of this protein restores the dendritic mode. By use of purified proteins, a small number of components are sufficient for the assembly of filopodia-like bundles: WASP-coated beads, actin, Arp2/3 complex, and fascin. On the basis of analysis of this system, we proposed a model for filopodial formation in which actin filaments of a preexisting dendritic network are elongated by inhibition of capping and subsequently cross-linked into bundles by fascin.

Digital fluorescence microscopy of cell cytoplasts with and without the centrosome

Publisher Summary This chapter describes the experimental protocols used for the preparation of samples for high-resolution digital fluorescence microscopy. For high-resolution digital fluorescence microscopy of microtubules (MTs), Cy3-tagged tubulin subunits are injected in living cells. Injected cells are treated with cytochalasin and nocodazole to disrupt actin filaments and MTs, respectively, and then enucleated by centrifugation. After washing out the drugs, images of fluorescently labeled microtubules in cytoplasts are captured with a cooled charge-coupled device (CCD) camera. In cytoplasts lacking the centrosome, decreased MT stability produces an elevated level of free tubulin and imaging of MTs is complicated by unusually high background fluorescence. Therefore, every effort must be made to improve the signal-to-noise ratio. A sensitive cooled CCD camera is used for capturing images. MTs are labeled with a very bright fluorophore to maximize the signal. Porcine brain tubulin subunits are conjugated with Cy3, which is significantly brighter than many other available fluorophores. The chapter describes the protocol used for the preparation of Cy3-tagged porcine brain tubulin subunits.

Microinjection of protein samples.

INTRODUCTIONThis protocol describes a method for microinjecting proteins into the nucleus or cytoplasm of adherent cells. Microinjection equipment can be obtained from a number of suppliers; this protocol has been used with the Narishige IM-200 air pressure regulator and the Leitz micromanipulator. Using this system, it is possible to microinject a constant volume within a 50% difference among cells.

Components of a Microinjection System

Direct-pressure microinjection with a micropipette is an essential tool for introducing a variety of impermeant substances into the cytoplasm or nucleus of plant and animal cells. Microinjection remains the most direct method to gain insight into the function and dynamics of intracellular components, to produce transgenic animals, or to overcome male infertility. This article describes the basic components of a microinjection system.

Calibration of Micropipette Tips

INTRODUCTIONThis protocol describes an easy method for calibrating micropipette tips that have been pulled in the laboratory. It is essential to estimate the internal diameter of the pulled micropipette tip when adjusting parameters for a new puller or new type of glass tubing. A tip diameter of ~0.3 μm is optimal for the microinjection of mammalian cells in culture (e.g., CHO, PtK1, and COS-7). A 10% increase in diameter increases the delivery rate by more than 30% and can cause cell damage. A smaller tip diameter will result in frequent clogging from protein aggregates.