SNARE proteins (soluble NSF-attachment protein receptors) are thought to be central components of the exocytotic mechanism in neurosecretory cells, but their precise function remained unclear. Here, we show that each of the vesicle-associated SNARE proteins (v-SNARE) of a chromaffin granule, synaptobrevin II or cellubrevin, is sufficient to support Ca 2 þ -dependent exocytosis and to establish a pool of primed, readily releasable vesicles. In the absence of both proteins, secretion is abolished, without affecting biogenesis or docking of granules indicating that v-SNAREs are absolutely required for granule exocytosis. We find that synaptobrevin II and cellubrevin differentially control the pool of readily releasable vesicles and show that the v-SNARE's amino terminus regulates the vesicle's primed state. We demonstrate that dynamics of fusion pore dilation are regulated by v-SNAREs, indicating their action throughout exocytosis from priming to fusion of vesicles.

Autophagy, a tightly regulated lysosome-dependent catabolic pathway, is important in the regulation of cancer development and progression and in determining the response of tumor cells to anticancer therapy. However, the role of autophagy in leukemia still remains largely unknown. Here we show that high-mobility group box 1 (HMGB1), the best characterized damage-associated molecular pattern, was released from leukemia cell lines after chemotherapy-induced cytotoxicity and activated autophagy to protect against injury. Treatment with HMGB1-neutralizing antibodies increased the sensitivity of leukemia cells to chemotherapy; whereas, exogenous HMGB1 rendered these cells more resistant to drug-induced cytotoxicity. Moreover, exogenous HMGB1 increased autophagy as evaluated by increased expression of the autophagic marker microtubule-associated protein light chain 3-II, degradation of sequestosome 1 (p62) and autophagosome formation. Furthermore, knockdown or pharmacological inhibition of either phosphoinositide 3-kinase-III or extracellular signal-regulated kinase kinase mitogen-activated protein kinase kinase/extracellular signal-regulated protein kinase inhibited HMGB1-induced autophagy. Taken together, these results suggest that HMGB1 release after chemotherapy is a critical regulator of autophagy and a potential drug target for therapeutic interventions in leukemia.

Neurotransmitter release is mediated by the SNARE proteins synaptobrevin II (sybII, also known as VAMP2), syntaxin, and SNAP-25, generating a force transfer to the membranes and inducing fusion pore formation. However, the molecular mechanism by which this force leads to opening of a fusion pore remains elusive. Here we show that the ability of sybII to support exocytosis is inhibited by addition of one or two residues to the sybII C terminus depending on their energy of transfer from water to the membrane interface, following a Boltzmann distribution. These results suggest that following stimulation, the SNARE complex pulls the C terminus of sybII deeper into the vesicle membrane. We propose that this movement disrupts the vesicular membrane continuity leading to fusion pore formation. In contrast to current models, the experiments suggest that fusion pore formation begins with molecular rearrangements at the intravesicular membrane leaflet and not between the apposed cytoplasmic leaflets.chromaffin cell | patch clamp capacitance measurement | caged calcium | amperometry | electrochemical detector array T he SNARE proteins (1) mediate release of stored secretory products by exocytosis. In neurosecretion, the t-SNAREs syntaxin and SNAP-25 in the plasma membrane bind the v-SNARE synaptobrevin II (sybII, also known as VAMP2), which is anchored to the vesicle membrane by a single transmembrane (TM) domain. Upon stimulation, the SNARE complex is thought to zip up more tightly proceeding in a vectorial manner from the N to the C terminus, toward the TM domains of sybII and syntaxin (2-5), thereby transferring a force to the membranes (6). However, the molecular mechanism by which this force leads to opening of the fusion pore has not been determined (7). Several models have been proposed to explain the mechanism of fusion pore formation. In the lipid-stalk-hemifusion hypothesis, the outer and the inner leaflets of the two membranes merge via formation of a hemifusion intermediate in response to forces exerted by proteins surrounding the fusion site (8). In an alternative proteinaceous fusion pore model, the fusion pore is lined by the TM domain of syntaxin (9) and possibly synaptobrevin (10). However, it is not immediately evident how the hydrophobic transmembrane domains can line an aqueous fusion pore that allows for ion permeation by electrodiffusion (11). When the C-terminal SNARE domain interactions are reduced by mutating or deleting the C terminus of SNAP-25, or when flexible linkers are introduced between the sybII TM domain and its SNARE domain, the rate of exocytosis is reduced (12-16) and the flux of transmitter through the early fusion pore is decreased (16,17), consistent with a structural change in the fusion pore. In an attempt to interpret these findings, a proteolipidic fusion pore model has been proposed, in which the fusion pore is formed by a molecular complex of both lipids and SNARE proteins (17). However, even this model does not explain the molecular mechanism by which the N-to C-terminal zipp...

Studies in mice have shown that both LT-and ST-HSCs lack an array of lineage markers (Lin Ϫ ) while simultaneously expressing stem-cell antigen-1 (Ly6A/E; Sca1) and high levels of the receptor tyrosine kinase, c-kit (CD117). 9,10 This phenotype has been abbreviated Lin Ϫ Sca1 ϩ c-kit Hi , or LSK. It constitutes only about 0.5% of whole bone marrow (WBM), but includes a heterogeneous population of HSCs and oligopotent progenitors. In mice expressing the Thy1.1 allele of CD90, HSCs are confined to the LSK subset expressing low levels of this marker (LSK-Thy1.1 Lo ). 11 This represents 0.15% of WBM, and 1 in 22 LSK-Thy1.1 Lo cells transplanted into radiation-conditioned hosts can concurrently repopulate B-cell, T-cell, and myeloid lineages. 12 However, ST multilineage repopulating cells within the LSK-Thy1.1 Lo compartment outnumber LT repopulating cells by 10 to 1, illustrating the paucity of LT-HSCs. Surface markers such as CD27, CD48, CD150, and Flt3/Flk2 (CD135) have been used in independent studies to further define and enrich LT-HSCs, as will be discussed in our results.The ability to identify distinct yet primitive hematopoietic progenitors has made it possible to question the molecular mechanisms governing differentiation between these compartments. The factors governing LT-to ST-HSC differentiation and how these differ from stimuli directing LT-HSC renewal are of special interest due to their potential therapeutic promises. However, characterizing molecular events in HSCs is not trivial due to the scarcity of these bone marrow progenitors and the labor-intensive protocols required to isolate them at high purities. Nevertheless, studies using genetic approaches are beginning to implicate several gene families as influential regulators of these processes. [13][14][15][16][17][18] Among these are the cell-cycle regulators p21 cip1/waf1 and p16 INK4a , which have been shown to influence LT-HSC maintenance by governing depletion of this compartment. [19][20][21] Helix-loop-helix (HLH) transcriptional regulators have also been shown to play a role in HSC maintenance. [22][23][24] These proteins can function as either transcriptional activators or repressors. E-proteins, encoded by E2A, HEB, and E2-2 genes, are a family of activators that may influence HSC maintenance by influencing transcription of p21 cip1/waf1 and p16 INK4a genes. [25][26][27][28] E-proteins are almost ubiquitously present; hence, their transcriptional activity is controlled by expression of one or more inhibitory proteins. One prominent class of E-protein inhibitors is the Id family, which includes Id1 through Id4. Id proteins have HLH domains similar to E-proteins, which allows avid binding to E-proteins to form inactive heterodimers. 29,30 Through this mechanism, Id proteins regulate a wide range of differentiation programs. For example, ectopic expression of Id1 from lymphoidspecific promoters abolishes B-or T-cell development, 31,32 and For personal use only. on May 10, 2018. by guest www.bloodjournal.org From overexpression in bone marr...

Vitamin D3 causes potent suppression of various cancer cells; however, significant supraphysiological concentrations of this compound are required for antineoplastic effects. Current combinatorial therapies with vitamin D3 are restricted to differentiation effects. It remains uncertain if autophagy is involved in vitamin D3 inhibition on leukemia cells. Here we show that besides triggering differentiation and inhibiting apoptosis, which was previously known, vitamin D3 triggers autophagic death in human myeloid leukemia cells. Inhibiting differentiation does not efficiently diminish vitamin D3 suppression on leukemia cells. Vitamin D3 up-regulates Beclin1, which binds to class III phosphatidylinositol 3-kinase to trigger autophagy. Vitamin D3 phosphorylates Bad in its BH3 domain, resulting in disassociation of the apoptotic Bad-Bcl-xL complex and association of Bcl-xL with Beclin1 and ultimate suppression of apoptotic signaling. Knockdown of Beclin1 eliminates vitamin D3-induced autophagy and inhibits differentiation but activates apoptosis, suggesting that Beclin1 is required for both autophagy and differentiation, and autophagy cooperates with differentiation but excludes apoptosis, in which Beclin1 acts as an interface for these three different cascades. Moreover, additional up-regulation of autophagy, but not apoptosis, dramatically improves vitamin D3 inhibition on leukemia cells. These findings extend our understanding of the action of vitamin D3 in antineoplastic effects and the role of Beclin1 in regulating multiple cellular cascades and suggest a potentially promising strategy with a significantly better antileukemia effect.1,25-Dihydroxyvitamin D3, the hormonally active form of vitamin D3, plays critical roles in regulating cellular and physiological responses. Treatment of vitamin D3 potently inhibits cell proliferation in a wide range of cancer cells, including myeloid leukemia and carcinomas of the breast, prostate, colon, skin, and brain and inhibits angiogenesis, tumor invasion, and metastases (1-8), suggesting that vitamin D3 has potential applications in cancer prevention and treatment. Vitamin D3 is known to inhibit cancer cell proliferation through the induction of differentiation, which is dependent on the signaling mechanisms involving down-regulation of Akt and its disassociation with Raf1 and subsequent activation of Raf/MEK/ERK 2

No abstract

The molecular mechanisms responsible for the 'distal' effect by which noradrenaline (NA) blocks exocytosis in the β-cell were examined by whole-cell and cell-attached patch clamp capacitance measurements in INS 832/13 β-cells. NA inhibited Ca Abbreviations BoNT/A, botulinum toxin A; Im, imaginary part of the patch admittance; mSIRK, myristoylated βγ-binding/activating peptide; NA, noradrenaline; PTX, pertussis toxin; Re, real part of the patch admittance; RRP, readily releasable pool; SNAP-25, synaptosomal-associated protein 25; SNARE, SNAP (soluble NSF attachment protein) receptors.

Transcriptional regulation is pivotal to the specification of distinct cell types during embryonic development. However, it still lacks a systematic way to identify key transcription factors (TFs) orchestrating the temporal and tissue specificity of gene expression. Here, we integrated epigenomic and transcriptomic data to reveal key regulators from two cells to postnatal day 0 in mouse embryogenesis. We predicted three-dimensional chromatin interactions in 12 tissues across eight developmental stages, which facilitates linking TFs to their target genes for constructing transcriptional regulatory networks. To identify driver TFs, we developed a new algorithm, dubbed Taiji, to assess the global influence of each TF and systematically uncovered TFs critical for lineage-specific and stage-dependent tissue specification. We have also identified TF combinations that function in spatiotemporal order to form transcriptional waves regulating developmental progress. Furthermore, lacking stage-specific TF combinations suggests a distributed timing strategy to orchestrate the coordination between tissues during embryonic development.