Dynamics of Immature Secretory Granules: Role of Cytoskeletal Elements during Transport, Cortical Restriction, and F-Actin-dependent Tethering

Secretory granules store neuropeptides and hormones and exhibit regulated exocytosis upon appropriate cellular stimulation. They are generated in the trans-Golgi network as immature secretory granules, short-lived vesicular intermediates, which undergo a complex and poorly understood maturation process. Due to their short half-life and low abundance, real-time studies of immature secretory granules have not been previously possible. We describe here a pulse/chase-like system based on the expression of a human chromogranin B-GFP fusion protein in neuroendocrine PC12 cells, which permits direct visualization of the budding of immature secretory granules and their dynamics during maturation. Live cell imaging revealed that newly formed immature secretory granules are transported in a direct and microtubule-dependent manner within a few seconds to the cell periphery. Our data suggest that the cooperative action of microtubules and actin filaments restricts immature secretory granules to the F-actin-rich cell cortex, where they move randomly and mature completely within a few hours. During this maturation period, secretory granules segregate into pools of different motility. In a late phase of maturation, 60% of secretory granules were found to be immobile and about half of these underwent F-actin-dependent tethering.

A Saccharomyces cerevisiae homolog of the human adrenoleukodystrophy transporter is a heterodimer of two half ATP-binding cassette transporters.

The adrenoleukodystrophy protein (ALDP) and the 70-kDa peroxisomal membrane protein (PMP70) are half ATP-binding cassette (ABC) transporters in the human peroxisome membrane. ALDP and PMP70 share sequence homology and both are implicated in genetic diseases. PXA1 and YKL741 are Saccharomyces cerevisiae genes that encode homologs of ALDP and PMP70. Pxa1p, a putative ortholog of ALDP, is involved in peroxisomal beta-oxidation of fatty acids while YKL741 is an open reading frame found by the yeast genome sequencing project. Here we designate YKL741 as PXA2 and show that its protein product, Pxa2p, like Pxa1p, is associated with peroxisomes but not required for their assembly. Yeast strains carrying gene disruption of PXA1, PXA2, or both have similar and, in the case of the latter, nonadditive phenotypes. We also find that the stability of Pxa1p, but not Pxa2p, is markedly reduced in the absence of the other. Finally, we find that Pxa1p and Pxa2p coimmuno-precipitate. These genetic and physical data suggest that Pxa1p and Pxa2p heterodimerize to form a complete peroxisomal ABC transporter involved in fatty acid beta-oxidation. This result predicts the presence of similar heterodimeric ABC transporters in the mammalian peroxisome membrane.

Half tetrad analysis in alfalfa using multiple restriction fragment length polymorphism markers.

A maximum likelihood approach of half tetrad analysis (HTA) based on multiple restriction fragment length polymorphism (RFLP) markers was developed. This procedure estimates the relative frequencies of 2n gametes produced by mechanisms genetically equivalent to first division restitution (FDR) or second division restitution and simultaneously locates the centromere within a linkage group of RFLP marker loci. The method was applied to the diploid alfalfa clone PG-F9 (2n = 2x = 16) previously selected because of its high frequency of 2n egg production. HTA was based on four RFLP loci for which PG-F9 was heterozygous with codominant alleles that were absent in the tetraploid tester. Models including three linked and one unlinked RFLP loci were developed and tested. Results of the HTA showed that PG-F9 produced 6% FDR and 94% second division restitution 2n eggs. Information from a marker locus belonging to one linkage group was used to more precisely locate the centromere on a different linkage group. HTA, together with previous cytological analysis, indicated that in PG-F9, FDR 2n eggs are likely produced by diplospory, a mechanism common among apomictic species. The occurrence of FDR 2n eggs in plant species and their importance for crop evolution and breeding is discussed together with the potential applicability of multilocus HTA in the study of reproductive mutants.

A candidate live inactivatable attenuated vaccine for AIDS.

The recent discovery of long term AIDS nonprogressors who harbor nef-attenuated HIV suggests that a naturally occurring live vaccine for AIDS may already exist. Animal models have shown that a live vaccine for AIDS, attenuated in nef, is the best candidate vaccine. There are considerable risks, real and perceived, with the use of live HIV vaccines. We have introduced a conditional lethal genetic element into HIV-1 and simian immunodeficiency virus (SIV) molecular clones deleted in nef. The antiviral strategy we employed targets both virus replication and the survival of the infected cell. The suicide gene, herpes simplex virus thymidine kinase (tk), was expressed and maintained in HIV over long periods of time. Herpes simplex virus tk confers sensitivity to the antiviral activity of acyclic nucleosides such as ganciclovir (GCV). HIV-tk and SIV-tk replication were sensitive to GCV at subtoxic concentrations, and virus-infected cells were eliminated from tumor cell lines as well as primary cell cultures. We found the HIV-tk virus to be remarkably stable even after being cultured in media containing a low concentration of GCV and then challenged with the higher dose and that while GCV resistant escape mutants did arise, a significant fraction of the virus remained sensitive to GCV.

Myosin dynamics in live Dictyostelium cells.

Conventional myosin plays a key role in the cytoskeletal reorganization necessary for cytokinesis, migration, and morphological changes associated with development in nonmuscle cells. We have made a fusion between the green fluorescent protein (GFP) and the Dictyostelium discoideum myosin heavy chain (GFP-myosin). The unique Dictyostelium system allows us to test the GFP-tagged myosin for activity both in vivo and in vitro. Expression of GFP-myosin rescues all myosin null cell defects. Additionally, GFP-myosin purified from these cells exhibits the same ATPase activities and in vitro motility as wild-type myosin. GFP-myosin is concentrated in the cleavage furrow during cytokinesis and in the posterior cortex of migrating cells. Surprisingly, GFP-myosin concentration increases transiently in the tips of retracting pseudopods. Contrary to previous thinking, this suggests that conventional myosin may play an important role in the dynamics of pseudopods as well as filopodia, lamellipodia, and other cellular protrusions.

Recombinant Listeria monocytogenes as a live vaccine vehicle for the induction of protective anti-viral cell-mediated immunity.

Listeria monocytogenes (LM) is a Gram-positive bacterium that is able to enter host cells, escape from the endocytic vesicle, multiply within the cytoplasm, and spread directly from cell to cell without encountering the extracellular milieu. The ability of LM to gain access to the host cell cytosol allows proteins secreted by the bacterium to efficiently enter the pathway for major histocompatibility complex class I antigen processing and presentation. We have established a genetic system for expression and secretion of foreign antigens by recombinant strains, based on stable site-specific integration of expression cassettes into the LM genome. The ability of LM recombinants to induce protective immunity against a heterologous pathogen was demonstrated with lymphocytic choriomeningitis virus (LCMV). LM strains expressing the entire LCMV nucleoprotein or an H-2Ld-restricted nucleoprotein epitope (aa 118-126) were constructed. Immunization of mice with LM vaccine strains conferred protection against challenge with virulent strains of LCMV that otherwise establish chronic infection in naive adult mice. In vivo depletion of CD8+ T cells from vaccinated mice abrogated their ability to clear viral infection, showing that protective anti-viral immunity was due to CD8+ T cells.