Sebastian Schuck

Resistance of cell membranes to different detergents

Partial resistance of cell membranes to solubilization with mild detergents and the analysis of isolated detergent-resistant membranes (DRMs) have been used operationally to define membrane domains. Given the multitude of detergents used for this purpose, we sought to investigate whether extraction with different detergents might reflect the same underlying principle of domain formation. We therefore compared the protein and lipid content of DRMs prepared with a variety of detergents from two cell lines. We found that the detergents differ considerably in their ability to selectively solubilize membrane proteins and to enrich sphingolipids and cholesterol over glycerophospholipids as well as saturated over unsaturated phosphatidylcholine. In addition, we observed cell type-dependent variations of the molecular characteristics of DRMs and the effectiveness of particular detergents. These results make it unlikely that different detergents reflect the same aspects of membrane organization and underscore both the structural complexity of cell membranes and the need for more sophisticated analytical tools to understand their architecture.

Polarized sorting in epithelial cells: raft clustering and the biogenesis of the apical membrane

Polarized cells establish and maintain functionally distinct surface domains by an elaborate sorting process, which ensures accurate delivery of biosynthetic cargo to different parts of the plasma membrane. This is particularly evident in polarized epithelial cells, which have been used as a model system for studies of sorting mechanisms. The clustering of lipid rafts through the oligomerization of raft components could be utilized for segregating apical from basolateral cargo and for the generation of intracellular transport carriers. Besides functioning in polarized sorting in differentiated cells, raft clustering might also play an important role in the biogenesis of apical membrane domains during development.

Membrane expansion alleviates endoplasmic reticulum stress independently of the unfolded protein response

Increasing the size of the ER by lipid synthesis helps the cell deal with ER stress.

ER-phagy: selective autophagy of the endoplasmic reticulum.

Throughout their life, cells must maintain homeostasis while facing constantly fluctuating demands on their different organelles. A major mechanism for the homeostatic control of organelle function is the unfolded protein response (UPR), a signaling pathway that triggers a comprehensive remodeling of the endoplasmic reticulum (ER) and the biosynthetic pathway according to need. We discovered that activation of the UPR in yeast also induces a new branch of macroautophagy that selectively targets the ER. We term this process “ER-phagy”, in analogy to pexophagy and mitophagy, the two other known forms of organelle-specific marcoautophagy. ER-phagy involves the generation of autophagosomes that selectively include ER membranes and whose delimiting double membranes also derive, at least in part, from the ER. This finding provides direct evidence that the ER can serve as a membrane source for autophagosome formation and indicates that ER-phagy entails engulfment of the ER by itself. ER-phagy could remove damaged or redundant parts of the ER and thus represent an important degradative functionality of the UPR that helps to afford homeostatic control. Addendum to: Autophagy Counterbalances Endoplasmic Reticulum Expansion during the Unfolded Protein Response S. Bernales, K.L. McDonald and P. Walter PLoS Biol 2006; 4: e423

Serum response factor is required for immediate-early gene activation yet is dispensable for proliferation of embryonic stem cells.

ABSTRACT Addition of serum to mitogen-starved cells activates the cellular immediate-early gene (IEG) response. Serum response factor (SRF) contributes to such mitogen-stimulated transcriptional induction of many IEGs during the G0-G1 cell cycle transition. SRF is also believed to be essential for cell cycle progression, as impairment of SRF activity by specific antisera or antisense RNA has previously been shown to block mammalian cell proliferation. In contrast, Srf −/− mouse embryos grow and develop up to E6.0. Using the embryonic stem (ES) cell system, we demonstrate here that wild-type ES cells do not undergo complete cell cycle arrest upon serum withdrawal but that they can mount an efficient IEG response. This IEG response, however, is severely impaired in Srf −/− ES cells, providing the first genetic proof that IEG activation is dependent upon SRF. Also, Srf−/− ES cells display altered cellular morphology, reduced cortical actin expression, and an impaired plating efficiency on gelatin. Yet, despite these defects, the proliferation rates of Srf −/− ES cells are not substantially altered, demonstrating that SRF function is not required for ES cell cycle progression.

Identification of T-Cell Antigens Specific for Latent Mycobacterium Tuberculosis Infection

Background T-cell responses against dormancy-, resuscitation-, and reactivation-associated antigens of Mycobacterium tuberculosis are candidate biomarkers of latent infection in humans. Methodology/Principal Findings We established an assay based on two rounds of in vitro restimulation and intracellular cytokine analysis that detects T-cell responses to antigens expressed during latent M. tuberculosis infection. Comparison between active pulmonary tuberculosis (TB) patients and healthy latently M. tuberculosis-infected donors (LTBI) revealed significantly higher T-cell responses against 7 of 35 tested M. tuberculosis latency-associated antigens in LTBI. Notably, T cells specific for Rv3407 were exclusively detected in LTBI but not in TB patients. The T-cell IFNγ response against Rv3407 in individual donors was the most influential factor in discrimination analysis that classified TB patients and LTBI with 83% accuracy using cross-validation. Rv3407 peptide pool stimulations revealed distinct candidate epitopes in four LTBI. Conclusions Our findings further support the hypothesis that the latency-associated antigens can be exploited as biomarkers for LTBI.

Homeostatic adaptation to endoplasmic reticulum stress depends on Ire1 kinase activity

Uncoupling of Ire1’s RNAse and kinase activities reveals that its auto-phosphorylation is important for resolution of the unfolded protein response. (See also a related paper by Chawla et al. in this issue).

Rab10 is Involved in Basolateral Transport in Polarized Madin–Darby Canine Kidney Cells

The sorting of newly synthesized membrane proteins to the cell surface is an important mechanism of cell polarity. To identify more of the molecular machinery involved, we investigated the function of the small GTPase Rab10 in polarized epithelial Madin–Darby canine kidney cells. We find that GFP‐tagged Rab10 localizes primarily to the Golgi during early cell polarization. Expression of an activated Rab10 mutant inhibits biosynthetic transport from the Golgi and missorts basolateral cargo to the apical membrane. Depletion of Rab10 by RNA interference has only mild effects on biosynthetic transport and epithelial polarization, but simultaneous inhibition of Rab10 and Rab8a more strongly impairs basolateral sorting. These results indicate that Rab10 functions in trafficking from the Golgi at early stages of epithelial polarization, is involved in biosynthetic transport to the basolateral membrane and may co‐operate with Rab8.

ER-phagy mediates selective degradation of endoplasmic reticulum independently of the core autophagy machinery.

ABSTRACT Selective autophagy of damaged or redundant organelles is an important mechanism for maintaining cell homeostasis. We found previously that endoplasmic reticulum (ER) stress in the yeast Saccharomyces cerevisiae causes massive ER expansion and triggers the formation of large ER whorls. Here, we show that stress-induced ER whorls are selectively taken up into the vacuole, the yeast lysosome, by a process termed ER-phagy. Import into the vacuole does not involve autophagosomes but occurs through invagination of the vacuolar membrane, indicating that ER-phagy is topologically equivalent to microautophagy. Even so, ER-phagy requires neither the core autophagy machinery nor several other proteins specifically implicated in microautophagy. Thus, autophagy of ER whorls represents a distinct type of organelle-selective autophagy. Finally, we provide evidence that ER-phagy degrades excess ER membrane, suggesting that it contributes to cell homeostasis by controlling organelle size.

Mycobacterium tuberculosis-specific CD4+, IFNγ+, and TNFα+ multifunctional memory T cells coexpress GM-CSF

Multifunctional T cells expressing several cytokines in parallel are thought to play a crucial role in protection against different infections. To characterize T cell cytokine patterns associated with disease and protection in Mycobacterium tuberculosis infection we determined the expression of IFNgamma, IL-2, TNFalpha, and GM-CSF in T cell subpopulations from children with tuberculosis (TB) and healthy latently M. tuberculosis-infected children (LTBI) after short-term in vitro restimulation. We identified CD4(+) effector memory T cells (T(EM)) as the major source of all measured cytokines after antigen-specific restimulation. T(EM) from children with TB expressed higher proportions of IFNgamma, TNFalpha, and IL-2 after Mtb restimulation while no differences were detected for GM-CSF between both study groups. GM-CSF secretion strongly depended on antigen-specific stimulation. Analyses of multiple cytokine patterns revealed that the majority of GM-CSF-positive M. tuberculosis-specific memory T cells coexpressed IFNgamma and TNFalpha therefore showing a characteristic feature of multifunctional T cells. We conclude that children with active TB possess higher proportions of IFNgamma-, TNFalpha-, and/or IL-2-positive T(EM) than children with LTBI while GM-CSF coexpression reveals a novel subpopulation within CD4(+) memory T cells not increased in children with active TB.