Crosslinking a lipid raft component triggers liquid ordered-liquid disordered phase separation in model plasma membranes.

The mechanisms by which a cell uses and adapts its functional membrane organization are poorly understood and are the subject of ongoing investigation and discussion. Here, we study one proposed mechanism: the crosslinking of membrane components. In immune cell signaling (and other membrane-associated processes), a small change in the clustering of specific membrane proteins can lead to large-scale reorganizations that involve numerous other membrane components. We have investigated the large-scale physical effect of crosslinking a minor membrane component, the ganglioside GM1, in simple lipid models of the plasma membrane containing sphingomyelin, cholesterol, and phosphatidylcholine. We observe that crosslinking GM1 can cause uniform membranes to phase-separate into large, coexistent liquid ordered and liquid disordered membrane domains. We also find that this lipid separation causes a dramatic redistribution of a transmembrane peptide, consistent with a raft model of membrane organization. These experiments demonstrate a mechanism that could contribute to the effects of crosslinking observed in cellular processes: Domains induced by clustering a small number of proteins or lipids might rapidly reorganize many other membrane proteins.

A role for actin, Cdc1p, and Myo2p in the inheritance of late Golgi elements in Saccharomyces cerevisiae.

In Saccharomyces cerevisiae, Golgi elements are present in the bud very early in the cell cycle. We have analyzed this Golgi inheritance process using fluorescence microscopy and genetics. In rapidly growing cells, late Golgi elements show an actin-dependent concentration at sites of polarized growth. Late Golgi elements are apparently transported into the bud along actin cables and are also retained in the bud by a mechanism that may involve actin. A visual screen for mutants defective in the inheritance of late Golgi elements yielded multiple alleles of CDC1. Mutations in CDC1 severely depolarize the actin cytoskeleton, and these mutations prevent late Golgi elements from being retained in the bud. The efficient localization of late Golgi elements to the bud requires the type V myosin Myo2p, further suggesting that actin plays a role in Golgi inheritance. Surprisingly, early and late Golgi elements are inherited by different pathways, with early Golgi elements localizing to the bud in a Cdc1p- and Myo2p-independent manner. We propose that early Golgi elements arise from ER membranes that are present in the bud. These two pathways of Golgi inheritance in S. cerevisiae resemble Golgi inheritance pathways in vertebrate cells.

Tagging Hansenula polymorpha genes by random integration of linear DNA fragments (RALF).

We have investigated the feasibility of using gene tagging by restriction enzyme-mediated integration (REMI) to isolate mutants in Hansenula polymorpha. A plasmid that cannot replicate in H. polymorpha and contains a dominant zeocin resistance cassette, pREMI-Z, was used as the integrative/mutagenic plasmid. We observed that high transformation efficiency was primarily dependent on the use of linearised pREMI-Z, and that the addition of restriction endonuclease to linearised pREMI-Z prior to transformation increased the transformation frequency only slightly. Integration of linearised pREMI-Z occurred at random in the H. polymorpha genome. Therefore, we termed this method Random integration of Linear DNA Fragments (RALF). To explore the potential of RALF in H. polymorpha, we screened a collection of pREMI-Z transformants for mutants affected in peroxisome biogenesis (pex) or selective peroxisome degradation (pdd). Many previously described PEX genes were obtained from the mutant collection, as well as a number of new genes, including H. polymorpha PEX12 and genes whose function in peroxisome biogenesis is still unclear. These results demonstrate that RALF is a powerful tool for tagging genes in H. polymorpha that should make it possible to carry out genome-wide mutagenesis screens.

Dynamics of transitional endoplasmic reticulum sites in vertebrate cells.

A typical vertebrate cell contains several hundred sites of transitional ER (tER). Presumably, tER sites generate elements of the ER-Golgi intermediate compartment (ERGIC), and ERGIC elements then generate Golgi cisternae. Therefore, characterizing the mechanisms that influence tER distribution may shed light on the dynamic behavior of the Golgi. We explored the properties of tER sites using Sec13 as a marker protein. Fluorescence microscopy confirmed that tER sites are long-lived ER subdomains. tER sites proliferate during interphase but lose Sec13 during mitosis. Unlike ERGIC elements, tER sites move very little. Nevertheless, when microtubules are depolymerized with nocodazole, tER sites redistribute rapidly to form clusters next to Golgi structures. Hence, tER sites have the unusual property of being immobile, yet dynamic. These findings can be explained by a model in which new tER sites are created by retrograde membrane traffic from the Golgi. We propose that the tER-Golgi system is organized by mutual feedback between these two compartments.

Raising the speed limits for 4D fluorescence microscopy.

Three-dimensional time-lapse (4D) fluorescence microscopy is becoming a routine experimental tool. This article summarizes current technologies, and describes a new method for speeding image acquisition during 4D confocal microscopy.

The Fanconi anemia group C gene product is located in both the nucleus and cytoplasm of human cells.

The Fanconi anemia (FA) complementation group C (FAC) protein gene encodes a cytoplasmic protein with a predicted Mr of 63,000. The protein's function is unknown, but it has been hypothesized that it either mediates resistance to DNA cross-linking agents or facilitates repair after exposure to such factors. The protein also plays a permissive role in the growth of colony-forming unit-granulocyte/macrophage (CFU-GM), burst-forming unit-erythroid (BFU-E), and CFU-erythroid (CFU-E). Attributing a specific function to this protein requires an understanding of its intracellular location. Recognizing that prior study has established the functional importance of its cytoplasmic location, we tested the hypothesis that FAC protein can also be found in the nucleus. Purified recombinant Escherichia coli-derived FAC antigens were used to create antisera able to specifically identify an Mr = 58,000 protein in lysates from human Epstein-Barr virus (EBV)-transformed cell lines by immunoblot analysis. Subcellular fractionation of the cell lysates followed by immunoblot analysis revealed that the majority of the FAC protein was cytoplasmic, as reported previously; however, approximately 10% of FAC protein was reproducibly detected in nuclear fractions. These results were reproducible by two different fractionation methods, and included markers to control for contamination of nuclear fractions by cytoplasmic proteins. Moreover, confocal image analysis of human 293 cells engineered to express FAC clearly demonstrated that FAC protein is located in both cytoplasmic and nuclear compartments, consistent with data obtained from fractionation of the FA cell lines. Finally, complementation of the FAC defect using retroviral-mediated gene transfer resulted in a substantial increase in nuclear FAC protein. Therefore, while cytoplasmic localization of this protein appears to be functionally important, it may also exert some essential nuclear function.

Potent effects of low levels of MHC class II-associated invariant chain on CD4+ T cell development.

Invariant chain (Ii)-negative mice exhibit defects in MHC class II assembly and transport that results in reduced levels of surface class II, altered antigen presentation, and inefficient positive selection of CD4+ T cells. Many CD4+ T cells that do mature in Ii-negative mice express a cell surface phenotype consistent with aberrant positive selection or peripheral activation. Reconstitution of these mice with low levels of either the p31 or p41 form of Ii does not restore transport of the bulk of class II or class II surface expression, but surprisingly does restore positive selection as measured by numbers and surface phenotype of CD4+ T cells. Thus, an Ii-dependent process, independent of effects on class II surface density, appears to be required for normal positive selection of CD4+ T cells.

The effect of low oxygen tension on the in vitro-replicative life span of human diploid fibroblast cells and their transformed derivatives.

Human diploid fibroblasts (HDF) IMR90, starting at various population doubling levels (PDL), were serially cultured under four different oxygen conditions; the conventional atmospheric 20% O2 condition and three lower oxygen conditions (1, 6, and 12% O2). All cultures from different PDLs showed that a longer replicative life span was achieved under the lower oxygen conditions. When the starting culture PDL was between 33 and 62, the increased life span rate was constant (average 22%) at 1% O2, but the rate decreased when the starting PDL was higher. The growth advantage under the 1% O2 condition was also observed in the very late passages of cultures to some extent, but terminal cultures with senescent cells were not stimulated by the low oxygen condition. When cultures at an extended PDL under the 1% O2 condition were shifted back to the 20% O2 condition, the cells rapidly senesced. HDF from a subject with Werner syndrome, a premature aging disease, which are known to have reduced replicative potential in vitro, also showed 43% increase in life span under the lowest oxygen conditions. When SV40 large T-transformed IMR90 cells at preimmortal stages were tested, no significant growth differences were observed under different oxygen conditions, and all the cultures died out at a similar PDL. These results suggest: (1) The atmospheric 20% oxygen tension hastens HDF senescence. (2) Young cells are more resistant to oxygen tension than old cells. (3) SV40 large T transformation abolishes the oxygen effect on cellular aging.

Hypersensitivity to oxygen is a uniform and secondary defect in Fanconi anemia cells.

Cells from patients with Fanconi anemia (FA) frequently show an increased sensitivity to DNA crosslinking agents such as mitomycin C (MMC). FA cells also show abnormal sensitivity to oxygen tension. In order to examine the correlation between the two cellular defects in FA, several FA fibroblast lines were tested for their sensitivity to MMC and oxygen by colony-formation frequency. The sensitivity to MMC in different FA lines varied in a broad range from normal level to extreme hypersensitivity, whereas all of the FA lines showed similar hypersensitivity to oxygen. When FA fibroblasts were transformed by SV40 large T-antigen, the hypersensitivity to oxygen was normalized while the MMC sensitivity still remained. These results suggest that the cellular sensitivity to oxygen is a secondary defect rather than a primary effect of mutations in FA. However, it is a more uniform phenotype than the MMC sensitivity, and therefore, it may be closely related to the common clinical symptoms of FA. Since 1% oxygen showed the highest colony-formation frequency for FA cells, establishment of FA primary fibroblasts was attempted at the low oxygen condition. FA fibroblast cells showed greatly enhanced growth and migration at 1% oxygen resulting in fast establishment of FA primary fibroblasts.