Multiple death signals influence mitochondria during apoptosis, yet the critical initiating event for mitochondrial dysfunction in vivo has been unclear. tBID, the caspase-activated form of a "BH3-domain-only" BCL-2 family member, triggers the homooligomerization of "multidomain" conserved proapoptotic family members BAK or BAX, resulting in the release of cytochrome c from mitochondria. We find that cells lacking both Bax and Bak, but not cells lacking only one of these components, are completely resistant to tBID-induced cytochrome c release and apoptosis. Moreover, doubly deficient cells are resistant to multiple apoptotic stimuli that act through disruption of mitochondrial function: staurosporine, ultraviolet radiation, growth factor deprivation, etoposide, and the endoplasmic reticulum stress stimuli thapsigargin and tunicamycin. Thus, activation of a "multidomain" proapoptotic member, BAX or BAK, appears to be an essential gateway to mitochondrial dysfunction required for cell death in response to diverse stimuli.

https://science.sciencemag.org/content/sci/292/5517/727.full.pdf

Proapoptotic BAX and BAK: A Requisite Gateway to Mitochondrial Dysfunction and Death

Vectors derived from human immunodeficiency virus (HIV) are highly efficient vehicles for in vivo gene delivery. However, their biosafety is of major concern. Here we exploit the complexity of the HIV genome to provide lentivirus vectors with novel biosafety features. In addition to the structural genes, HIV contains two regulatory genes, tat and rev, that are essential for HIV replication, and four accessory genes that encode critical virulence factors. We previously reported that the HIV type 1 accessory open reading frames are dispensable for efficient gene transduction by a lentivirus vector. We now demonstrate that the requirement for the tat gene can be offset by placing constitutive promoters upstream of the vector transcript. Vectors generated from constructs containing such a chimeric long terminal repeat (LTR) transduced neurons in vivo at very high efficiency, whether or not they were produced in the presence of Tat. When the rev gene was also deleted from the packaging construct, expression of gag and pol was strictly dependent on Rev complementation in trans. By the combined use of a separate nonoverlapping Rev expression plasmid and a 5' LTR chimeric transfer construct, we achieved optimal yields of vector of high transducing efficiency (up to 10(7) transducing units [TU]/ml and 10(4) TU/ng of p24). This third-generation lentivirus vector uses only a fractional set of HIV genes: gag, pol, and rev. Moreover, the HIV-derived constructs, and any recombinant between them, are contingent on upstream elements and trans complementation for expression and thus are nonfunctional outside of the vector producer cells. This split-genome, conditional packaging system is based on existing viral sequences and acts as a built-in device against the generation of productive recombinants. While the actual biosafety of the vector will ultimately be proven in vivo, the improved design presented here should facilitate testing of lentivirus vectors.

A Third-Generation Lentivirus Vector with a Conditional Packaging System

The morphology and cytoskeletal structure of fibroblasts, endothelial cells, and neutrophils are documented for cells cultured on surfaces with stiffness ranging from 2 to 55,000 Pa that have been laminated with fibronectin or collagen as adhesive ligand. When grown in sparse culture with no cell-cell contacts, fibroblasts and endothelial cells show an abrupt change in spread area that occurs at a stiffness range around 3,000 Pa. No actin stress fibers are seen in fibroblasts on soft surfaces, and the appearance of stress fibers is abrupt and complete at a stiffness range coincident with that at which they spread. Upregulation of 5 integrin also occurs in the same stiffness range, but exogenous expression of 5 integrin is not sufficient to cause cell spreading on soft surfaces. Neutrophils, in contrast, show no dependence of either resting shape or ability to spread after activation when cultured on surfaces as soft as 2 Pa compared to glass. The shape and cytoskeletal differences evident in single cells on soft compared to hard substrates are eliminated when fibroblasts or endothelial cells make cell-cell contact. These results support the hypothesis that mechanical factors impact different cell types in fundamentally different ways, and can trigger specific changes similar to those stimulated by soluble ligands. Cell Motil. Cytoskeleton 60:24 –34, 2005. © 2004 Wiley-Liss, Inc.

/pdf/effects-of-substrate-stiffness-on-cell-morphology-59za0lba3e.pdf

Effects of substrate stiffness on cell morphology, cytoskeletal structure, and adhesion

Morphogenesis and oncogenesis of MCF-10A mammary epithelial acini grown in three-dimensional basement membrane cultures

Generation of human induced pluripotent stem cells by direct delivery of reprogramming proteins.

We have generated a human 293-derived retroviral packaging cell line (293GPG) capable of producing high titers of recombinant Moloney murine leukemia virus particles that have incorporated the vesicular stomatitis virus G (VSV-G) protein. To achieve expression of the retroviral gag-pol polyprotein, the precise coding sequences for gag-pol were introduced into a vector which utilizes totally nonretroviral signals for gene expression. Because constitutive expression of the VSV-G protein is toxic in 293 cells, we used the tetR/VP 16 transactivator and teto minimal promoter system for inducible, tetracycline-regulatable expression of VSV-G. After stable transfection of the 293GPG packaging cell line with the MFG.SnlsLacZ retroviral vector construct, it was possible to readily isolate stable virus-producing cell lines with titers approaching 10(7) colony-forming units/ml. Transient transfection of 293GPG cells using a modified version of MFG.SnlsLacZ, in which the cytomegalovirus IE promoter was used to drive transcription of the proviral genome, led to titers of approximately 10(6) colony-forming units/ml. The retroviral/VSV-G pseudotypes generated using 293GPG cells were significantly more resistant to human complement than commonly used amphotropic vectors and could be highly concentrated (> 1000-fold). This new packaging cell line may prove to be particularly useful for assessing the potential use of retroviral vectors for direct in vivo gene transfer. The design of the cell line also provides at least theoretical advantages over existing cell lines with regard to the possible release of replication-competent virus.

https://www.pnas.org/content/pnas/93/21/11400.full.pdf

A stable human-derived packaging cell line for production of high titer retrovirus/vesicular stomatitis virus G pseudotypes

Mutualistic interactions benefit both partners, promoting coexistence and genetic diversity. Spatial structure can promote cooperation, but spatial expansions may also make it hard for mutualistic partners to stay together, because genetic drift at the expansion front creates regions of low genetic and species diversity. To explore the antagonism between mutualism and genetic drift, we grew cross-feeding strains of the budding yeast Saccharomyces cerevisiae on agar surfaces as a model for mutualists undergoing spatial expansions. By supplying varying amounts of the exchanged nutrients, we tuned strength and symmetry of the mutualistic interaction. Strong mutualism suppresses genetic demixing during spatial expansions and thereby maintains diversity, but weak or asymmetric mutualism is overwhelmed by genetic drift even when mutualism is still beneficial, slowing growth and reducing diversity. Theoretical modeling using experimentally measured parameters predicts the size of demixed regions and how strong mutualism must be to survive a spatial expansion.

/pdf/genetic-drift-opposes-mutualism-during-spatial-population-jr5dlj8e3d.pdf

Genetic drift opposes mutualism during spatial population expansion

How locally adapted ecotypes are established and maintained within a species is a long-standing question in evolutionary biology. Using forest and prairie ecotypes of deer mice (Peromyscus maniculatus), we characterized the genetic basis of variation in two defining traits—tail length and coat color—and discovered a 41-megabase chromosomal inversion linked to both. The inversion frequency is 90% in the dark, long-tailed forest ecotype; decreases across a habitat transition; and is absent from the light, short-tailed prairie ecotype. We implicate divergent selection in maintaining the inversion at frequencies observed in the wild, despite high levels of gene flow, and explore fitness benefits that arise from suppressed recombination within the inversion. We uncover a key role for a large, previously uncharacterized inversion in the evolution and maintenance of classic mammalian ecotypes. Description Maintaining difference Species often comprise several ecotypes, distinct populations that occupy different habitats. Ecotypes can persist over long time periods, even with substantial gene flow between them, which raises the question of how they maintain their locally adaptive phenotypes over time. Hager et al. examined the genetic basis of two traits, tail length and coat color, that define the forest and prairie ecotypes of deer mice. They found a large chromosomal inversion that links redder coats and longer tails in the forest ecotype. Modeling suggests that the inversion originated under divergent selection many thousands of generations ago and likely provided a benefit to the forest ecotype by suppressing recombination despite gene flow. —BEL A large chromosomal inversion under divergent selection maintains distinct forest and prairie ecotypes of deer mice.

A chromosomal inversion contributes to divergence in multiple traits between deer mouse ecotypes

Mutualistic interactions benefit both partners, promoting coexistence and genetic diversity. Spatial structure can promote cooperation, but spatial expansions may also make it hard for mutualistic partners to stay together, since genetic drift at the expansion front creates regions of low genetic and species diversity. To explore the antagonism between mutualism and genetic drift, we grew cross-feeding strains of the budding yeast S. cerevisiae on agar surfaces as a model for mutualists undergoing spatial expansions. By supplying varying amounts of the exchanged nutrients, we tuned strength and symmetry of the mutualistic interaction. Strong mutualism suppresses genetic demixing during spatial expansions and thereby maintains diversity, but weak or asymmetric mutualism is overwhelmed by genetic drift even when mutualism is still beneficial, slowing growth and reducing diversity. Theoretical modeling using experimentally measured parameters predicts the size of demixed regions and how strong mutualism must be to survive a spatial expansion.

https://www.biorxiv.org/content/biorxiv/early/2014/02/25/003012.full.pdf

Beverly I. Neugeboren

Papers

A stable human-derived packaging cell line for production of high titer retrovirus/vesicular stomatitis virus G pseudotypes

Genetic drift opposes mutualism during spatial population expansion

A chromosomal inversion contributes to divergence in multiple traits between deer mouse ecotypes

Genetic drift opposes mutualism during spatial population expansion