Blair Bowers

Effect of ADP and Ionic Strength on the Kinetic and Motile Properties of Recombinant Mouse Myosin V

Mouse myosin V is a two-headed unconventional myosin with an extended neck that binds six calmodulins. Double-headed (heavy meromyosin-like) and single-headed (subfragment 1-like) fragments of mouse myosin V were expressed in Sf9 cells, and intact myosin V was purified from mouse brain. The actin-activated MgATPase of the tissue-purified myosin V, and its expressed fragments had a high V max and a lowK ATPase. Calcium regulated the MgATPase of intact myosin V but not of the fragments. Both the MgATPase activity and the in vitro motility were remarkably insensitive to ionic strength. Myosin V and its fragments translocated actin at very low myosin surface densities. ADP markedly inhibited the actin-activated MgATPase activity and the in vitromotility. ADP dissociated from myosin V subfragment 1 at a rate of about 11.5 s−1 under conditions where theV max was 3.3 s−1, indicating that, although not totally rate-limiting, ADP dissociation was close to the rate-limiting step. The high affinity for actin and the slow rate of ADP release helps the myosin head to remain attached to actin for a large fraction of each ATPase cycle and allows actin filaments to be moved by only a few myosin V molecules in vitro.

DEVELOPMENTALLY REGULATED EXPRESSION OF A GIARDIA LAMBLIA CYST WALL PROTEIN GENE

The protozoan Giardia lamblia is an obligate parasite of the mammalian small intestine. We studied the expression of a gene that encodes a protein component of the cyst wall, a complex structure assembled during the differentiation of trophozoites to cysts and which is critical to survival of the parasite outside its mammalian host. Transcripts from the cyst wall protein gene increase more than 100‐fold during encystation, reaching a maximum between 5 and 24 hours after induction. Cyst wall protein expression also increases dramatically during encystation, and, prior to its incorporation into the nascent cyst wall, the protein is contained within the encystation‐specific vesicles of encysting trophozoites. The sequence of the cloned gene predicts an acidic, leucine‐rich polypeptide of Mr, 26000 that contains 5.3 tandemly arranged copies of a degenerate 24‐amino‐acid repeat. A hydrophobic amino‐terminal peptide probably serves as the initial signal that targets this protein to a secretory pathway involving vesicular localization during encystation and, ultimately, secretion to form the cyst wall.

A Molecular Model for Morphogenesis: The Primary Septum of Yeast

Publisher Summary This chapter discusses a molecular model for morphogenesis. The simplest explanation of the asymmetry that results from cell separation is that the mother cell retains both the primary and secondary septa that go to form the bud scar; the daughter cell retains only a secondary septum, the birth scar. If indeed chitin constitutes the primary septum, it should be synthesized during a specific period of the cell cycle, that is, prior to cell division. To determine whether this was the case, a synchronous culture of Saccharomyces carlsbergensis was allowed to grow in a medium containing tritiated glucose, and the incorporation into chitin was monitored. Both the polysaccharide label and the number of cells increased in stepwise fashion whereas the total radioactivity in the cells increased exponentially. Polyoxins are a group of peptidyl-pyrimidine nucleoside antibiotics from Streptomyces cacaoi that are active against a variety of fungi. It was considered that their site of action was related to the biosynthesis of cell wall chitin.

Rho1p mutations specific for regulation of β(1→3)glucan synthesis and the order of assembly of the yeast cell wall

In the yeast Saccharomyces cerevisiae, the GTP-binding protein Rho1 is required for beta(1-->3)glucan synthase activity, for activation of protein kinase C and the cell integrity pathway and for progression in G1, cell polarization and exocytosis. A genetic screen for cells that become permeabilized at non-permissive temperature was used to isolate in vitro-generated mutants of Rho1p. After undergoing a battery of tests, several of them appeared to be specifically defective in the beta(1-->3) glucan synthesis function of Rho1p. At the non-permissive temperature (37 degrees C), the mutants developed defects in the cell wall, especially at the tip of new buds. In the yeast cell wall, beta(1-->6)glucan is linked to both beta(1-->3)glucan and mannoprotein, as well as occasionally to chitin. We have used the rho1 mutants to study the order of assembly of the cell wall components. The incorporation of [(14)C]-glucose into beta(1-->3)glucan at 37 degrees C was decreased or abolished in the mutants. Concomitantly, a partial defect in the incorporation of label into cell wall mannoproteins and beta(1-->6)glucan was observed. In contrast, YW3458, an inhibitor of glycosylphosphatidylinositol anchor formation, prevented mannoprotein incorporation, whereas the beta(1-->3)-beta(1-->6)glucan complex was synthesized at almost normal levels. As beta(1-->3)glucan can be synthesized in vitro or in vivo independently, we conclude that the order of addition in vivo is beta(1-->3)glucan, beta(1-->6)glucan, mannoprotein. Previous observations indicate that chitin is the last component to be incorporated into the complex.

Chitin Biosynthesis and Morphogenetic Processes

Chitin, one of the most abundant substances of biological origin, is an important component of cell walls and septa of many fungi (Wessels and Sietsma 1981; Ruiz-Herrera 1992). Because walls and septa have a well-defined shape and an essential role in cell maintenance and growth, there has been increasing interest in chitin biogenesis. Thus, the synthesis of chitin in fungi has served as a useful model for morphogenesis (Cabib 1987; Cabib et al. 1982b, 1988; Bulawa 1993).

Co-assembly of Envoplakin and Periplakin into Oligomers and Ca2+-dependent Vesicle Binding IMPLICATIONS FOR CORNIFIED CELL ENVELOPE FORMATION IN STRATIFIED SQUAMOUS EPITHELIA

Plakin family members envoplakin and periplakin have been shown to be part of the cornified cell envelope in terminally differentiating stratified squamous epithelia. In the present study, purified recombinant human envoplakin and periplakin were used to investigate their properties and interactions. We found that envoplakin was insoluble at physiological conditions in vitro, and co-assembly with periplakin was required for its solubility. Envoplakin and periplakin formed soluble complexes with equimolar stoichiometry. Chemical cross-linking revealed that the major soluble form of all periplakin constructs and of envoplakin/periplakin rod domains was a dimer, although co-assembly of the full-length proteins resulted in formation of higher order oligomers. Electron microscopy of rotary-shadowed periplakin demonstrated thin flexible molecules with an average contour length of 88 nm for the rod-plus-tail fragment, and immunolabeling EM confirmed the molecule as a parallel, in-register, dimer. Both periplakin and envoplakin/periplakin oligomers were able to bind synthetic lipid vesicles whose composition mimicked the cytoplasmic side of the plasma membrane of eukaryotic cells. This binding was dependent on anionic phospholipids and Ca2+. These findings raise the possibility that envoplakin and periplakin bind to the plasma membrane upon elevation of intracellular [Ca2+] in differentiating keratinocytes, where they serve as a scaffold for cornified cell envelope assembly.

Rho1p mutations specific for regulation of β(1→3)glucan synthesis and the order of assembly of the yeast cell wall: Rho1p mutants

In the yeast Saccharomyces cerevisiae, the GTP‐binding protein Rho1 is required for β(1→3)glucan synthase activity, for activation of protein kinase C and the cell integrity pathway and for progression in G1, cell polarization and exocytosis. A genetic screen for cells that become permeabilized at non‐permissive temperature was used to isolate in vitro‐generated mutants of Rho1p. After undergoing a battery of tests, several of them appeared to be specifically defective in the β(1→3)glucan synthesis function of Rho1p. At the non‐permissive temperature (37°C), the mutants developed defects in the cell wall, especially at the tip of new buds. In the yeast cell wall, β(1→6)glucan is linked to both β(1→3)glucan and mannoprotein, as well as occasionally to chitin. We have used the rho1 mutants to study the order of assembly of the cell wall components. The incorporation of [14C]‐glucose into β(1→3)glucan at 37°C was decreased or abolished in the mutants. Concomitantly, a partial defect in the incorporation of label into cell wall mannoproteins and β(1→6)glucan was observed. In contrast, YW3458, an inhibitor of glycosylphosphatidylinositol anchor formation, prevented mannoprotein incorporation, whereas the β(1→3)–β(1→6)glucan complex was synthesized at almost normal levels. As β(1→3)glucan can be synthesized in vitro or in vivo independently, we conclude that the order of addition in vivo is β(1→3)glucan, β(1→6)glucan, mannoprotein. Previous observations indicate that chitin is the last component to be incorporated into the complex.

Carmil is a bonafide capping protein interactant

CARMIL, also known as Acan 125, is a multidomain protein that was originally identified on the basis of its interaction with the Src homology 3 (SH3) domain of type I myosins from Acanthamoeba. In a subsequent study of CARMIL from Dictyostelium, pull-down assays indicated that the protein also bound capping protein and the Arp2/3 complex. Here we present biochemical evidence that Acanthamoeba CARMIL interacts tightly with capping protein. In biochemical preparations, CARMIL copurified extensively with two polypeptides that were shown by microsequencing to be the α- and β-subunits of Acanthamoeba capping protein. The complex between CARMIL and capping protein, which is readily demonstratable by chemical cross-linking, can be completely dissociated by size exclusion chromatography at pH 5.4. Analytical ultracentrifugation, surface plasmon resonance and SH3 domain pull-down assays indicate that the dissociation constant of capping protein for CARMIL is ∼0.4 μm or lower. Using CARMIL fusion proteins, the binding site for capping protein was shown to reside within the carboxyl-terminal, ∼200 residue, proline-rich domain of CARMIL. Finally, chemical cross-linking, analytical ultracentrifugation, and rotary shadowed electron microscopy revealed that CARMIL is asymmetric and that it exists in a monomer ↔ dimer equilibrium with an association constant of 1.0 × 106 m-1. Together, these results indicate that CARMIL self-associates and interacts with capping protein with affinities that, given the cellular concentrations of the proteins (∼1 and 2 μm for capping protein and CARMIL, respectively), indicate that both activities should be physiologically relevant.

Carbohydrates as structural constituents of yeast cell wall and septum

The cell walls and septa of fungi a re useful models for morphogenesis and potential targets for anti fungal agents. In the yeast, Saccharomvces cerevisiae, the main structural components of the primary septum and of the cell wall are chitin and ,¶(1+3)glucan, respectively. Two chitin synthetases have been identified in yeast, one of which is essential for septum formation, whereas the other one has a repair function. in extracts in a zymogenic form that can be activated by proteases. /?(1-+3)Glucan synthetase is also attached to the plasma membrane. activity is strongly stimulated by GTP and its analogs. extraction of the membranes, two components have been obtained

Localization of acid phosphatase in Acanthamoeba castellanii with light and electron microscopy during growth and after phagocytosis.

By use of light and electron microscopes the localization of acid phosphatase was determined by cytochemical reaction in Acanthamoeba castellanii (Neff strain), during axenic growth and during phagocytosis. The cell density of the cultures was found to have a marked effect on the ease of cytochemical demonstration of acid phosphatase. During axenic growth, this enzyme was found in about half of the cytoplasmic vacuoles, in some vesicles of the Golgi apparatus, and in mitochondria-like structures in the cytoplasm. A quantitative study made during phagocytosis of the changes in number of acid phosphatase-positive vacuoles and -positive phagosomes gave new data on the digestive system of this amoeba and on its behavior during axenic growth and during phagocytosis. Biochemical assays indicated no induction of acid phosphatase by phagocytosis and no external release of hydrolase during phagocytosis.