Characterizing the Transport and Surface Affinity of Extracellular Vesicles Isolated from Yeast and Bacteria in Well-Characterized Porous Media.

Extracellular vesicles (EVs) are membrane-bounded, nanosized particles, produced and secreted by all biological cell types. EVs are ubiquitous in the environment, operating in various roles including intercellular communication and plant immune modulation. Despite their ubiquity, the role of EV surface chemistry in determining transport has been minimally investigated. Using the zeta (ζ)-potential as a surrogate for surface charge, this work considers the deposition of EVs from the yeast, Saccharomyces cerevisiae, and two bacterial species, Staphylococcus aureus and Pseudomonas fluorescens, in well-characterized porous medium under various background conditions shown to influence the transport of other environmental colloidal particles: ionic strength and humic acid concentration. The affinity of S. cerevisiae EVs for the porous medium (glass beads) appeared to be sensitive to changes in ionic strength, as predicted by colloid stability (Derjaguin, Landau, Verwey, and Overbeek or DLVO) theory, and humic acid concentration, while P. fluorescens EVs deviated from DLVO predictions, suggesting that mechanisms other than charge stabilization may control the deposition of P. fluorescens. Calculations of attachment efficiency from these deposition studies were used to estimate EV transport using a clean-bed filtration model. Based on these calculations, EVs could be transported through such homogeneous porous media up to 15 m.

The Gα-interacting vesicle-associated protein interacts with and promotes cell surface localization of GRP78 during endoplasmic reticulum stress.

Cell surface translocation of the chaperone glucose-regulated protein 78 kDa (GRP78) is a key event that promotes cancer cell survival during endoplasmic reticulum (ER) stress. Here, we identify Gα-interacting vesicle-associated protein (GIV) - an enhancer of prosurvival signaling during ER stress - as a binding partner of GRP78. We show that GIV and GRP78 interact in an ER stress-dependent manner through their respective carboxyl terminal domains and that GIV aids in the localization of GRP78 to the plasma membrane. Kaplan-Meier analysis of disease-free survival in cancer patients shows poor prognosis for patients with high expression of both GIV and GRP78, further suggesting a vital role for these two proteins in enhancing cancer cell viability.

Identification and characterization of a novel phosphoregulatory site on cyclin-dependent kinase 5.

Cyclin-dependent kinase 5 (CDK5) is a serine/threonine kinase essential for embryonic development whose overactivation has been implicated in several pathologies including neurodegeneration, cancer cell metastasis and type II diabetes. Therefore, it is important to investigate molecular mechanism(s) that mediate regulation of CDK5 activity. Here we identify and characterize a novel phosphoregulatory site on CDK5. Our mass spectrometry analysis identified seven putative phosphorylation sites on CDK5. Using phosphomimetic and non-phosphorylatable mutants, we determined that phosphorylation of S47, one of the identified sites, renders the kinase catalytically inactive. The inactivation of the kinase due to the phosphomimetic change at S47 results from inhibition of its interaction with its cognate activator, p35. We connect the effect of this regulatory event to a cellular phenotype by showing that the S47D CDK5 mutant inhibits cell migration and promotes cell proliferation. Together, these results have uncovered a potential physiological mechanism to regulate CDK5 activity. The evolutionary placement of a phosphorylatable residue (S/T) at this position not only in CDK5 but also in the majority of other CDK family members suggests that this phosphosite may represent a shared regulatory mechanism across the CDK family.

Genetically Programmed Changes in Photosynthetic Cofactor Metabolism in Copper-deficient Chlamydomonas.

Genetic and genomic studies indicate that copper deficiency triggers changes in the expression of genes encoding key enzymes in various chloroplast-localized lipid/pigment biosynthetic pathways. Among these are CGL78 involved in chlorophyll biosynthesis and HPPD1, encoding 4-hydroxyphenylpyruvate dioxygenase catalyzing the committed step of plastoquinone and tocopherol biosyntheses. Copper deficiency in wild-type cells does not change the chlorophyll content, but a survey of chlorophyll protein accumulation in this situation revealed increased accumulation of LHCSR3, which is blocked at the level of mRNA accumulation when either CGL78 expression is reduced or in the crd1 mutant, which has a copper-nutrition conditional defect at the same step in chlorophyll biosynthesis. Again, like copper-deficient crd1 strains, cgl78 knock-down lines also have reduced chlorophyll content concomitant with loss of PSI-LHCI super-complexes and reduced abundance of a chlorophyll binding subunit of PSI, PSAK, which connects LHCI to PSI. For HPPD1, increased mRNA results in increased abundance of the corresponding protein in copper-deficient cells concomitant with CRR1-dependent increased accumulation of γ-tocopherols, but not plastoquinone-9 nor total tocopherols. In crr1 mutants, where increased HPPD1 expression is blocked, plastochromanol-8, derived from plastoquinone-9 and purported to also have an antioxidant function, is found instead. Although not previously found in algae, this metabolite may occur only in stress conditions.