Membrane proximal lysosomes are the major vesicles responsible for calcium-dependent exocytosis in nonsecretory cells

Similar to its role in secretory cells, calcium triggers exocytosis in nonsecretory cells. This calcium-dependent exocytosis is essential for repair of membrane ruptures. Using total internal reflection fluorescence microscopy, we observed that many organelles implicated in this process, including ER, post-Golgi vesicles, late endosomes, early endosomes, and lysosomes, were within 100 nm of the plasma membrane (in the evanescent field). However, an increase in cytosolic calcium led to exocytosis of only the lysosomes. The lysosomes that fused were predominantly predocked at the plasma membrane, indicating that calcium is primarily responsible for fusion and not recruitment of lysosomes to the cell surface

Imaging single events at the cell membrane

The ability to sense and respond to the environment is a hallmark of living systems. These processes occur at the levels of the organism, cells and individual molecules. Sensing of extracellular changes could result in a structural or chemical alteration in a molecule, which could in turn trigger a cascade of intracellular signals or regulated trafficking of molecules at the cell surface. These and other such processes allow cells to sense and respond to environmental changes. Often, these changes and the responses to them are spatially and/or temporally localized, and visualization of such events necessitates the use of highresolution imaging approaches. Here we discuss optical imaging approaches and tools for imaging individual events at the cell surface with improved speed and resolution. The use of ensemble measurements, which report changes that affect an averaged outcome, has been crucial in our understanding of transmembrane signaling. Such studies have identified various cell surface receptors and signaling mechanisms that detect and respond to extracellular signals, such as nutrients, growth factors, pathogens and the extracellular matrix. However, many physiological and pathological events affect individual molecules or organelles. Because information is lost by averaging signals, determining important mechanistic details necessitates studying individual events. For example, if a microscopic event exists in two or more states, such as a signaling cascade that is either on or off or an ion channel that is open or closed, the average will represent a state that does not exist at the microscopic level; averaging the amplitude of response thus results in a loss of information about individual events. As a second example, ensemble measurements report the dominant population but miss responses that occur from a minority of spatially or temporally localized signals. A third example is the inability to temporally order single events on the basis of an averaged measurement. Ordering the steps of a multistep process by ensemble study requires the individual events to be tightly synchronized, which is difficult to achieve and even more difficult to maintain. However, observing individual events allows the order of each step to be unambiguously determined without a need for synchronization. A fourth example is the loss of important temporal information about individual events in ensemble averages. Each molecule might alter its state exponentially over time, or the change might be abrupt but the distribution of when the change occurs among individual molecules might be exponential. The temporal nature of such microscopic events often cannot be resolved from ensemble measurements. Many approaches have been developed to improve the spatial and temporal resolution with which we can study cellular and molecular responses at or near the cell surface. Approaches such as electron microscopy allow very high spatial resolution but are not suitable for imaging dynamic processes. Other approaches, such as scanning probe microscopy (which includes atomic force microscopy) and near-field scanning optical microscopy, offer spatial resolution at the nanometer scale. However, when scanning an area even as big as the surface of a cell, the temporal resolution becomes poor. Further, these methods do not allow simultaneous measurement of activities all across the cell surface, thus limiting their applicability for monitoring cell surface events in real time. The use of electrical measurements, such as voltage-clamp recording, allows membrane events to be studied at submillisecond temporal resolution. However, when performed at the whole-cell level, electrophysiology does not provide spatial information. Information on finer spatial detail can be gathered with patch-clamping to study single channels and transporters. However, this allows sampling at only a single spot. Since the introduction of patch-clamping three decades ago 1 , it has been applied widely to study cell surface processes. Excellent discussion of these approaches, their applications and comparison with optical imaging are available 2-5 and will not be discussed here. Many of the limits on spatial and temporal resolution can be overcome by optical imaging. Optical imaging permits monitoring from molecular to organismal scales and for time periods ranging from milliseconds to several days. In the first part of this review, we will discuss developments that have improved the speed and resolution of live cell imaging. In the second part, we will highlight the contributions and practical applications of these techniques to improve monitoring of single events at the cell membrane. Tools for optimizing spatial and temporal resolution Conventional optical microscopy allows an axial resolution of approximately 400 nm to be achieved. However, the cell membrane is two orders of magnitude smaller, at a thickness of approximately 4-6 nm (refs. 6-8) Several approaches allow the reduction or elimination of out-ofplane fluorescence, and a few of these can surmount the physical limit imposed by the wavelength of visible ligh

Transcriptional profiling of putative human epithelial stem cells

<p>Abstract</p> <p>Background</p> <p>Human interfollicular epidermis is sustained by the proliferation of stem cells and their progeny, transient amplifying cells. Molecular characterization of these two cell populations is essential for better understanding of self renewal, differentiation and mechanisms of skin pathogenesis. The purpose of this study was to obtain gene expression profiles of alpha 6⁺/MHCI⁺, transient amplifying cells and alpha 6⁺/MHCI^-, putative stem cells, and to compare them with existing data bases of gene expression profiles of hair follicle stem cells. The expression of Major Histocompatibility Complex (MHC) class I, previously shown to be absent in stem cells in several tissues, and alpha 6 integrin were used to isolate MHCI positive basal cells, and MHCI low/negative basal cells.</p> <p>Results</p> <p>Transcriptional profiles of the two cell populations were determined and comparisons made with published data for hair follicle stem cell gene expression profiles. We demonstrate that presumptive interfollicular stem cells, alpha 6⁺/MHCI^-cells, are enriched in messenger RNAs encoding surface receptors, cell adhesion molecules, extracellular matrix proteins, transcripts encoding members of IFN-alpha family proteins and components of IFN signaling, but contain lower levels of transcripts encoding proteins which take part in energy metabolism, cell cycle, ribosome biosynthesis, splicing, protein translation, degradation, DNA replication, repair, and chromosome remodeling. Furthermore, our data indicate that the cell signaling pathways Notch1 and NF-κB are downregulated/inhibited in MHC negative basal cells.</p> <p>Conclusion</p> <p>This study demonstrates that alpha 6⁺/MHCI^-cells have additional characteristics attributed to stem cells. Moreover, the transcription profile of alpha 6⁺/MHCI^-cells shows similarities to transcription profiles of mouse hair follicle bulge cells known to be enriched for stem cells. Collectively, our data suggests that alpha 6⁺/MHCI^-cells may be enriched for stem cells. This study is the first comprehensive gene expression profile of putative human epithelial stem cells and their progeny that were isolated directly from neonatal foreskin tissue. Our study is important for understanding self renewal and differentiation of epidermal stem cells, and for elucidating signaling pathways involved in those processes. The generated data base may serve those working with other human epithelial tissue progenitors.</p

VHL Type 2B gene mutation moderates HIF dosage in vitro and in vivo

Von Hippel-Lindau (VHL) disease is caused by germline mutations in the VHL tumor suppressor gene, with Type 2B missense VHL mutations predisposing to renal cell carcinoma, hemangioblastoma, and pheochromocytoma. Type 2B mutant pVHL is predicted to be defective in hypoxia inducible factor (HIF)-α regulation. Murine embryonic stem (ES) cells in which the endogenous wild-type Vhl gene was replaced with the representative Type 2B VHL hotspot mutation R167Q (Vhl2B/2B) displayed preserved physiologic regulation of both HIF factors with slightly more normoxic dysregulation of HIF-2α. Differentiated Vhl2B/2B-derived teratomas over-expressed the joint HIF targets Vegf and EglN3 but not the HIF-1α-specific target Pfk1 and displayed a growth advantage over Vhl-/--derived teratomas, suggestive of a tight connection between perturbations in the degree and ratio of HIF-1α and HIF-2α stabilization and cell growth. Vhl2B/2B mice displayed mid-gestational embryonic lethality, while adult Vhl2B/+ mice exhibited susceptibility to carcinogen-promoted renal neoplasia compared with wild-type littermates at twelve months. Our experiments support a model in which the representative Type 2B R167Q mutant pVhl produces a unique profile of HIF dysregulation, thereby promoting tissue-specific effects on cell growth, development, and tumor predisposition

Differential Regulation of Lipoprotein and Hepatitis C Virus Secretion by Rab1b

Secretory cells produce diverse cargoes, yet how they regulate concomitant secretory traffic remains insufficiently explored. Rab GTPases control intracellular vesicular transport. To map secretion pathways, we generated a library of lentivirus-expressed dominant-negative Rab mutants and used it in a large-scale screen to identify regulators of hepatic lipoprotein secretion. We identified several candidate pathways, including those mediated by Rab11 and Rab8. Surprisingly, inhibition of Rab1b, the major regulator of transport from the endoplasmic reticulum to the Golgi, differently affected the secretion of the very-low-density lipoprotein components ApoE and ApoB100, despite their final association on mature secreted lipoprotein particles. Since hepatitis C virus (HCV) incorporates ApoE and ApoB100 into its virus particle, we also investigated infectious HCV secretion and show that its regulation by Rab1b mirrors that of ApoB100. These observations reveal differential regulation of hepatocyte secretion by Rab1b and advance our understanding of lipoprotein assembly and lipoprotein and HCV secretion

Patients and animal models of CNGβ1-deficient retinitis pigmentosa support gene augmentation approach.

Retinitis pigmentosa (RP) is a major cause of blindness that affects 1.5 million people worldwide. Mutations in cyclic nucleotide-gated channel β 1 (CNGB1) cause approximately 4% of autosomal recessive RP. Gene augmentation therapy shows promise for treating inherited retinal degenerations; however, relevant animal models and biomarkers of progression in patients with RP are needed to assess therapeutic outcomes. Here, we evaluated RP patients with CNGB1 mutations for potential biomarkers of progression and compared human phenotypes with those of mouse and dog models of the disease. Additionally, we used gene augmentation therapy in a CNGβ1-deficient dog model to evaluate potential translation to patients. CNGB1-deficient RP patients and mouse and dog models had a similar phenotype characterized by early loss of rod function and slow rod photoreceptor loss with a secondary decline in cone function. Advanced imaging showed promise for evaluating RP progression in human patients, and gene augmentation using adeno-associated virus vectors robustly sustained the rescue of rod function and preserved retinal structure in the dog model. Together, our results reveal an early loss of rod function in CNGB1-deficient patients and a wide window for therapeutic intervention. Moreover, the identification of potential biomarkers of outcome measures, availability of relevant animal models, and robust functional rescue from gene augmentation therapy support future work to move CNGB1-RP therapies toward clinical trials