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Hippocampal neurons in tissue culture develop functional synapses that exhibit considerable variation in synaptic vesicle content (20–350 vesicles). We examined absolute and fractional parameters of synaptic vesicle exocytosis of individual synapses. Their correlation to vesicle content was determined by activity-dependent discharge of FM-styryl dyes. At high frequency stimulation (30 Hz), synapses with large recycling pools released higher amounts of dye, but showed a lower fractional release compared to synapses that contained fewer vesicles. This effect gradually vanished at lower frequencies when stimulation was triggered at 20 Hz and 10 Hz, respectively. Live-cell antibody staining with anti-synaptotagmin-1-cypHer 5, and overexpression of synaptopHluorin as well as photoconversion of FM 1-43 followed by electron microscopy, consolidated the findings obtained with FM-styryl dyes. We found that the readily releasable pool grew with a power function with a coefficient of 2/3, possibly indicating a synaptic volume/surface dependency. This observation could be explained by assigning the rate-limiting factor for vesicle exocytosis at high frequency stimulation to the available active zone surface that is proportionally smaller in synapses with larger volumes.

Objective:Associations between body mass index (BMI) and mental disorders meeting Axis‐I diagnoses according to the Diagnostic and Statistical Manual for Mental Disorders IV (DSM‐IV) were investigated in The Early Developmental Stages of Psychopathology Study in a large population‐based sample, which included adolescents and young adults of both genders for the first time. Research Methods and Procedures:A total of 3021 German subjects ranging from 14 to 24 years of age were assessed for specific DSM‐IV diagnoses derived from a modified version of the standardized Composite International Diagnostic Interview, and general psychological disturbances, using the Symptom Checklist‐90‐Revised. BMI percentiles for age and gender were calculated to avoid systematic bias in the BMI distribution resulting from the young age range represented in the sample. Additionally, subjects with a lifetime diagnosis of any eating disorder were excluded from statistical analysis to control the confounding effect of body weight‐related eating disorders on associations between BMI and psychopathology. Results:The results based on logistic regression analyses and MANOVAs demonstrate that the BMI is not associated with mental disorders or general psychopathologies. There were no significant associations between BMI and mood, anxiety, substance, and somatoform disorders, a result that contrasts with almost all previous clinical studies. Additionally, in contrast to clinical investigations and most epidemiological studies, neither obesity nor underweight was significantly associated with any kind of general psychopathology. Discussion:The overall finding that obesity is not significantly related to marked psychopathology in the general German population of adolescents and young adults has important clinical implications.

Exocytosis and ‘kiss-and-run’ secretion coexist in chromaffin cells. Our findings suggest that these mechanisms are closely related, based on their common molecular machinery. Here we present a model that describes how chromaffin cells regulate catecholamine release by switching the mode of secretion between the two pathways, a process controlled by phosphorylation. Stimulation-dependent vesicle-plasma membrane interactions in chromaffin cells were analysed by simultaneous ‘on-cell’ capacitance and conductance measurements, a technique that allows the monitoring of single vesicles. Capacitance steps represent fusions of large dense-core vesicles with the plasma membrane, whereas capacitance flickers correspond to transient connections of the vesicle lumen with the extracellular space. All these events require the presence of extracellular calcium in millimolar concentrations. ‘Kiss-and-run’ type of release is enhanced by the kinase inhibitor staurosporine, which suggests that this secretion mode is regulated by protein phosphorylation. We also observed capacitance bursts, which most probably represent ‘hot spots’ of secretion and we found that ‘kiss-and-run’ is the prevalent mechanism during these episodes. The significance of ‘kiss-and run’ for neurohormone release is even higher at physiological temperature, because up to half of all secretion events are mediated by this mechanism.

This study was focussed on the identification of the endocytic organelles in chromaffin cells which retrieve large, dense core vesicle (LDCV)‐membrane components from the plasma membrane. For this purpose, ‘on‐cell’ capacitance measurements and electron microscopy were employed. We found capacitance steps and capacitance flickers, corresponding to single exo‐ and endocytic events. The analysis revealed that the total membrane surface of completely fused LDCVs is recycled by large endocytic vesicles and smaller, most likely clathrin‐coated vesicles, at approximately the same ratio. These results were confirmed by rapid‐freeze immuno‐electron microscopy, where an extracellular marker was rapidly internalized into endocytic vesicles that morphologically resembled LDCVs.

This study was focussed on the identification of the endocytic organelles in chromaffin cells which retrieve large, dense core vesicle (LDCV)-membrane components from the plasma membrane. For this purpose, ‘on-cell’ capacitance measurements and electron microscopy were employed. We found capacitance steps and capacitance flickers, corresponding to single exo- and endocytic events. The analysis revealed that the total membrane surface of completely fused LDCVs is recycled by large endocytic vesicles and smaller, most likely clathrin-coated vesicles, at approximately the same ratio. These results were confirmed by rapid-freeze immuno-electron microscopy, where an extracellular marker was rapidly internalized into endocytic vesicles that morphologically resembled LDCVs.

Dictyostelium discoideummyosin Ik (MyoK) is a novel type of myosin distinguished by a remarkable architecture. MyoK is related to class I myosins but lacks a cargo-binding tail domain and carries an insertion in a surface loop suggested to modulate motor velocity. This insertion shows similarity to a secondary actin-binding site present in the tail of some class I myosins, and indeed a GST-loop construct binds actin. Probably as a consequence, binding of MyoK to actin was not only ATP-but also salt-dependent. Moreover, as both binding sites reside within its motor domain and carry potential sites of regulation, MyoK might represent a new form of actin crosslinker. MyoK was distributed in the cytoplasm with a significant enrichment in dynamic regions of the cortex. Absence of MyoK resulted in a drop of cortical tension whereas overexpression led to significantly increased tension. Absence and overexpression of MyoK dramatically affected the cortical actin cytoskeleton and resulted in reduced initial rates of phagocytosis. Cells lacking MyoK showed excessive ruffling, mostly in the form of large lamellipodia, accompanied by a thicker basal actin cortex. At early stages of development, aggregation of myoKnull cells was slowed due to reduced motility. Altogether, the data indicate a distinctive role for MyoK in the maintenance and dynamics of the cell cortex.

Kex2 in the yeast Saccharomyces cerevisiae is a transmembrane, Ca2+-dependent serine protease of the subtilisin-like pro-protein convertase (SPC) family with specificity for cleavage after paired basic amino acids. At steady state, Kex2 is predominantly localized in late Golgi compartments and initiates the proteolytic maturation of pro-protein precursors that transit the distal secretory pathway. However, Kex2 localization is not static, and its itinerary apparently involves transiting out of the late Golgi and cycling back from post-Golgi endosomal compartments during its lifetime. We tested whether the endocytic pathway could deliver small molecules to Kex2 from the extracellular medium. Here we report that intramolecularly quenched fluorogenic substrates taken up into intact yeast revealed fluorescence due to specific cleavage by Kex2 protease in endosomal compartments. Furthermore, the endocytic delivery of protease inhibitors interfered with Kex2 activity for precursor protein processing. These observations reveal that the endocytic pathway does intersect with the cycling itinerary of active Kex2 protease. This strategy of endocytic drug delivery has implications for modulating SPC protease activity needed for hormone, toxin and viral glycoprotein precursor processing in human cells.