We have identified a novel protein, p22, required for "constitutive" exocytic membrane traffic. p22 belongs to the EF-hand superfamily of Ca2+-binding proteins and shows extensive similarity to the regulatory subunit of protein phosphatase 2B, calcineurin B. p22 is a cytosolic N-myristoylated protein that undergoes conformational changes upon binding of Ca2+. Antibodies against a p22 peptide block the targeting/fusion of transcytotic vesicles with the apical plasma membrane, but recombinant wild-type p22 overcomes that inhibition. Nonmyristoylated p22, or p22 incapable of undergoing Ca2+-induced conformational changes, cannot reverse the antibody-mediated inhibition. The data suggest that p22 may act by transducing cellular Ca2+ signals to downstream effectors. p22 is ubiquitously expressed, and we propose that its function is required for membrane trafficking events common to many cells.

EnvZ, a membrane receptor kinase-phosphatase, modulates porin expression in Escherichia coli in response to medium osmolarity. It shares its basic scheme of signal transduction with many other sensor-kinases, passing information from the amino-terminal, periplasmic, sensory domain via the transmembrane helices to the carboxy-terminal, cytoplasmic, catalytic domain. The native receptor can exist in two active but opposed signaling states, the OmpR kinase-dominant state (K+ P−) and the OmpR-P phosphatase-dominant state (K− P+). The balance between the two states determines the level of intracellular OmpR-P, which in turn determines the level of porin gene transcription. To study the structural requirements for these two states of EnvZ, mutational analysis was performed. Mutations that preferentially affect either the kinase or phosphatase have been identified and characterized both in vivo and in vitro. Most of these mapped to previously identified structural motifs, suggesting an important function for each of these conserved regions. In addition, we identified a novel motif that is weakly conserved among two-component sensors. Mutations that alter this motif, which is termed the X region, alter the confirmation of EnvZ and significantly reduce the phosphatase activity.

Proteins containing the EF-hand Ca(2+)-binding motif, such as calmodulin and calcineurin B, function as regulators of various cellular processes. Here we focus on p22, an N-myristoylated, widely expressed EF-hand Ca(2+)-binding protein conserved throughout evolution, which was shown previously to be required for membrane traffic. Immunofluorescence studies show that p22 distributes along microtubules during interphase and mitosis in various cell lines. Moreover, we report that p22 associates with the microtubule cytoskeleton indirectly via a cytosolic microtubule-binding factor. Gel filtration studies indicate that the p22-microtubule-binding activity behaves as a 70- to 30-kDa globular protein. Our results indicate that p22 associates with microtubules via a novel N-myristoylation-dependent mechanism that does not involve classic microtubule-associated proteins and motor proteins. The association of p22 with microtubules requires the N-myristoylation of p22 but does not involve p22's Ca(2+)-binding activity, suggesting that the p22-microtubule association and the role of p22 in membrane traffic are functionally related, because N-myristoylation is required for both events. Therefore, p22 is an excellent candidate for a protein that can mediate interactions between the microtubule cytoskeleton and membrane traffic.

Many studies have used velocity measurements, waveform analyses, and theoretical flagella models to investigate the establishment, maintenance, and function of flagella of the biflagellate green algae Chlamydomonas reinhardtii. We report the first direct measurement of Chlamydomonas flagellar swimming force. Using an optical trap ("optical tweezers") we detect a 75% decrease in swimming force between wild type (CC124) cells and mutants lacking outer flagellar dynein arms (oda1). This difference is consistent with previous estimates and validates the force measurement approach. To examine mechanisms underlying flagella organization and function, we deflagellated cells and examined force generation during flagellar regeneration. As expected, fully regenerated flagella are functionally equivalent to flagella of untreated wild type cells. However, analysis of swimming force vs. flagella length and the increase in force over regeneration time reveals intriguing patterns where increases in force do not always correspond with increases in length. These investigations of flagellar force, therefore, contribute to the understanding of Chlamydomonas motility, describe phenomena surrounding flagella regeneration, and demonstrate the advantages of the optical trapping technique in studies of cell motility.

Respiratory health is negatively impacted by exposure to ozone or to estrogens. Increasingly, individuals have simultaneous environmental exposure to both compounds. Characterizing the cellular responses stimulated by the combination of ozone and estrogens, therefore, is crucial to our complete understanding of the compounds' environmental health impacts. Our work introduces an alveolar cell culture model with defined media that provides evidence of ozone damage and determines sex hormones alter the cells' susceptibility to oxidative damage. Specifically, we investigated the individual and combined effects of environmentally relevant levels of ozone and 17β-estradiol on non-cancerous rat, type-II alveolar cells by examining biomarkers of cellular health and redox balance. The data reveal a complex role for 17β-estradiol in cellular recovery from 1 hr exposure to high ozone levels. At 0.5 hr post-ozone necrosis and inflammation markers show 17β-estradiol augments the detrimental effects of 350 ppb ozone, but after 24 hr of recovery, steroid treatment alters glutathione redox ratio and allows cellular proliferation.