Beclin 1 regulates growth factor receptor signaling in breast cancer.

Beclin 1 is a haploinsufficient tumor suppressor that is decreased in many human tumors. The function of beclin 1 in cancer has been attributed primarily to its role in the degradative process of macroautophagy. However, beclin 1 is a core component of the vacuolar protein sorting 34 (Vps34)/class III phosphatidylinositoI-3 kinase (PI3KC3) and Vps15/p150 complex that regulates multiple membrane-trafficking events. In the current study, we describe an alternative mechanism of action for beclin 1 in breast cancer involving its control of growth factor receptor signaling. We identify a specific stage of early endosome maturation that is regulated by beclin 1, the transition of APPL1-containing phosphatidyIinositol 3-phosphate-negative (PI3P(-)) endosomes to PI3P(+) endosomes. Beclin 1 regulates PI3P production in response to growth factor stimulation to control the residency time of growth factor receptors in the PI3P(-)/APPL(+)-signaling-competent compartment. As a result, suppression of BECN1 sustains growth factor-stimulated AKT and ERK activation resulting in increased breast carcinoma cell invasion. In human breast tumors, beclin 1 expression is inversely correlated with AKT and ERK phosphorylation. Our data identify a novel role for beclin 1 in regulating growth factor signaling and reveal a mechanism by which loss of beclin 1 expression would enhance breast cancer progression.

The fleet feet of haematopoietic stem cells: rapid motility, interaction and proteopodia.

Haematopoietic stem cells (HSCs) have been extensively characterized regarding in vivo engraftment, surface epitopes and genetic regulation. However, little is known about the homing of these rare cells, and their intrinsic motility and membrane deformation capacity. We used high-speed optical-sectioning microscopy and inverted fluorescent videomicroscopy to study highly purified murine lineage-negative, rhodamine-low, Hoechst-low HSCs over time under various in vitro conditions. We discovered extremely rapid motility, directed migration to stromal cells and marked membrane modulation. High resolution images with three-dimensional reconstruction showed the general presence of microspikes. Further, pseudopodia (proteopodia) were observed that were induced by stromal-derived factor-1 and steel factor. Proteopodia were directed towards and were quenched by stromal cells, at times bridged HSCs, and could rapidly retract or detach from cells. Proteopodia were also observed in vivo with homed HSCs in frozen sections of murine spleen, lung and heart. This is the first demonstration that HSCs are both fast and highly malleable in phenotype.

Intercellular calcium waves in HeLa cells expressing GFP-labeled connexin 43, 32, or 26.

This study was undertaken to obtain direct evidence for the involvement of gap junctions in the propagation of intercellular Ca(2+) waves. Gap junction-deficient HeLa cells were transfected with plasmids encoding for green fluorescent protein (GFP) fused to the cytoplasmic carboxyl termini of connexin 43 (Cx43), 32 (Cx32), or 26 (Cx26). The subsequently expressed GFP-labeled gap junctions rendered the cells dye- and electrically coupled and were detected at the plasma membranes at points of contact between adjacent cells. To correlate the distribution of gap junctions with the changes in [Ca(2+)](i) associated with Ca(2+) waves and the distribution of the endoplasmic reticulum (ER), cells were loaded with fluorescent Ca(2+)-sensitive (fluo-3 and fura-2) and ER membrane (ER-Tracker) dyes. Digital high-speed microscopy was used to collect a series of image slices from which the three-dimensional distribution of the gap junctions and ER were reconstructed. Subsequently, intercellular Ca(2+) waves were induced in these cells by mechanical stimulation with or without extracellular apyrase, an ATP-degrading enzyme. In untransfected HeLa cells and in the absence of apyrase, cell-to-cell propagating [Ca(2+)](i) changes were characterized by initiating Ca(2+) puffs associated with the perinuclear ER. By contrast, in Cx-GFP-transfected cells and in the presence of apyrase, [Ca(2+)](i) changes were propagated without initiating perinuclear Ca(2+) puffs and were communicated between cells at the sites of the Cx-GFP gap junctions. The efficiency of Cx expression determined the extent of Ca(2+) wave propagation. These results demonstrate that intercellular Ca(2+) waves may be propagated simultaneously via an extracellular pathway and an intracellular pathway through gap junctions and that one form of communication may mask the other.

Microtubules regulate local Ca2+ spiking in secretory epithelial cells.

The role of the cytoskeleton in regulating Ca(2+) release has been explored in epithelial cells. Trains of local Ca(2+) spikes were elicited in pancreatic acinar cells by infusion of inositol trisphosphate through a whole cell patch pipette, and the Ca(2+)-dependent Cl(-) current spikes were recorded. The spikes were only transiently inhibited by cytochalasin B, an agent that acts on microfilaments. In contrast, nocodazole (5-100 micrometer), an agent that disrupts the microtubular network, dose-dependently reduced spike frequency and decreased spike amplitude leading to total blockade of the response. Consistent with an effect of microtubular disruption, colchicine also inhibited spiking but neither Me(2)SO nor beta-lumicolchicine, an inactive analogue of colchicine, had any effect. The microtubule-stabilizing agent, taxol, also inhibited spiking. The nocodazole effects were not due to complete loss of function of the Ca(2+) signaling apparatus, because supramaximal carbachol concentrations were still able to mobilize a Ca(2+) response. Finally, as visualized by 2-photon excitation microscopy of ER-Tracker, nocodazole promoted a loss of the endoplasmic reticulum in the secretory pole region. We conclude that microtubules specifically maintain localized Ca(2+) spikes at least in part because of the local positioning of the endoplasmic reticulum.

Close contacts with the endoplasmic reticulum as determinants of mitochondrial Ca2+ responses.

The spatial relation between mitochondria and endoplasmic reticulum (ER) in living HeLa cells was analyzed at high resolution in three dimensions with two differently colored, specifically targeted green fluorescent proteins. Numerous close contacts were observed between these organelles, and mitochondria in situ formed a largely interconnected, dynamic network. A Ca2+-sensitive photoprotein targeted to the outer face of the inner mitochondrial membrane showed that, upon opening of the inositol 1,4,5-triphosphate (IP3)-gated channels of the ER, the mitochondrial surface was exposed to a higher concentration of Ca2+ than was the bulk cytosol. These results emphasize the importance of cell architecture and the distribution of organelles in regulation of Ca2+ signaling.

Metabolic modulation of hexokinase association with mitochondria in living smooth muscle cells.

Hexokinase isoform I binds to mitochondria of many cell types. It has been hypothesized that this association is regulated by changes in the concentrations of specific cellular metabolites. To study the distribution of hexokinase in living cells, fluorophore-labeled functional hexokinase I was prepared. After microinjection into A7r5 smooth muscle cells, hexokinase localized to distinct structures identified as mitochondria. The endogenous hexokinase demonstrated a similar distribution with the use of immunocytochemistry. 2-Deoxyglucose elicited an increase in glucose 6-phosphate (G-6-P) and a decrease in ATP levels and diminished hexokinase binding to mitochondria in single cells. 3-O-methylglucose elicited slowly developing decreases in all three parameters. In contrast, cyanide elicited a rapid decrease in both ATP and hexokinase binding. Analyses of changes in metabolite levels and hexokinase binding indicate a positive correlation between binding and cell energy state as monitored by ATP. On the other hand, only in the presence of 2-deoxyglucose was the predicted inverse correlation between binding and G-6-P observed. Unlike the relatively large changes in distribution observed with the fluorescent-injected hexokinase, cyanide caused only a small decrease in the localization of endogenous hexokinase with mitochondria. These findings suggest that changes in the concentrations of specific metabolites can alter the binding of hexokinase I to specific sites on mitochondria. Moreover, the apparent difference in sensitivity of injected and endogenous hexokinase to changes in metabolites may reflect the presence of at least two classes of binding mechanisms for hexokinase, with differential sensitivity to metabolites.

Coupling of the Na+/Ca2+ exchanger, Na+/K+ pump and sarcoplasmic reticulum in smooth muscle.

The Na+/Ca2+ exchanger, driven by a transmembrane Na+ gradient, plays a key role in regulating Ca2+ concentration in many cells. Although the exchanger influences Ca2+ concentration, its activity in smooth muscle appears to be closely coupled to Ca2+ availability from intracellular stores. This linkage might result if the exchanger were positioned close to Ca2+ storage sites within the sarcoplasmic reticulum. To test this hypothesis we have developed methods to assess the relative three-dimensional distribution of proteins involved in Na+/K+ pumping, Na+/Ca2+ exchange, Ca2+ storage within the sarcoplasmic reticulum, and attachment of contractile filaments to the membrane in smooth muscle. Here we report that the Na+/Ca2+ exchanger is largely co-distributed with the Na+/K+ pump on unique regions of the plasma membrane in register with, and close to, calsequestrin-containing regions of the sarcoplasmic reticulum in sites distinct from the sites where contractile filaments attach to the membrane. This molecular organization suggests that the plasma membrane is divided into at least two functional domains, and appear to provide a mechanism for the strong linkage seen in smooth muscle between Na+/K+ pumping and Na+/Ca2+ exchange, and between Na+/Ca2+ exchange and Ca2+ release from the sarcoplasmic reticulum.