Clathrin mediates membrane fission and budding by constricting membrane pores.

Membrane budding, which underlies fundamental processes like endocytosis, intracellular trafficking, and viral infection, is thought to involve membrane coat-forming proteins, including the most observed clathrin, to form Ω-shape profiles and helix-forming proteins like dynamin to constrict Ω-profiles' pores and thus mediate fission. Challenging this fundamental concept, we report that polymerized clathrin is required for Ω-profiles' pore closure and that clathrin around Ω-profiles' base/pore region mediates pore constriction/closure in neuroendocrine chromaffin cells. Mathematical modeling suggests that clathrin polymerization at Ω-profiles' base/pore region generates forces from its intrinsically curved shape to constrict/close the pore. This new fission function may exert broader impacts than clathrin's well-known coat-forming function during clathrin (coat)-dependent endocytosis, because it underlies not only clathrin (coat)-dependent endocytosis, but also diverse endocytic modes, including ultrafast, fast, slow, bulk, and overshoot endocytosis previously considered clathrin (coat)-independent in chromaffin cells. It mediates kiss-and-run fusion (fusion pore closure) previously considered bona fide clathrin-independent, and limits the vesicular content release rate. Furthermore, analogous to results in chromaffin cells, we found that clathrin is essential for fast and slow endocytosis at hippocampal synapses where clathrin was previously considered dispensable, suggesting clathrin in mediating synaptic vesicle endocytosis and fission. These results suggest that clathrin and likely other intrinsically curved coat proteins are a new class of fission proteins underlying vesicle budding and fusion. The half-a-century concept and studies that attribute vesicle-coat contents' function to Ω-profile formation and classify budding as coat-protein (e.g., clathrin)-dependent or -independent may need to be re-defined and re-examined by considering clathrin's pivotal role in pore constriction/closure.

Multiple Modes of Fusion and Retrieval at the Calyx of Held Synapse.

Neurotransmitter in vesicles is released through a fusion pore when vesicles fuse with the plasma membrane. Subsequent retrieval of the fused vesicle membrane is the key step in recycling exocytosed vesicles. Application of advanced electrophysiological techniques to a large nerve terminal, the calyx of Held, has led to recordings of endocytosis, individual vesicle fusion and retrieval, and the kinetics of the fusion pore opening process and the fission pore closure process. These studies have revealed three kinetically different forms of endocytosis-rapid, slow, and bulk-and two forms of fusion-full collapse and kiss-and-run. Calcium influx triggers all kinetically distinguishable forms of endocytosis at calyces by activation of calmodulin/calcineurin signaling pathway and protein kinase C, which may dephosphorylate and phosphorylate endocytic proteins. Polymerized actin may provide mechanical forces to bend the membrane, forming membrane pits, the precursor for generating vesicles. These research advancements are reviewed in this chapter.

Light and electron microscopic imaging of synaptic vesicle endocytosis at mouse hippocampal cultures.

Following the release of neurotransmitters at synaptic vesicles via exocytosis, endocytosis is initiated to retrieve vesicles that have fused with the plasma membrane of nerve terminals and recycle them, thus sustaining synaptic transmission. Here, we describe imaging-based protocols for quantitative measurements of endocytosis at cultured synapses. These protocols include (1) primary culture of mouse hippocampal neurons, (2) studying endocytosis at neurons transfected with a pH-sensitive synaptophysin-pHluorin2× using fluorescent microscopy, and (3) imaging endocytosis at fixed neurons with electron microscopy. For complete details on the use and execution of this protocol, please refer to Wu et al. (2016) and Wu et al. (2021).

Presynaptic Kv3 channels are required for fast and slow endocytosis of synaptic vesicles.

Since their discovery decades ago, the primary physiological and pathological effects of potassium channels have been attributed to their ion conductance, which sets membrane potential and repolarizes action potentials. For example, Kv3 family channels regulate neurotransmitter release by repolarizing action potentials. Here we report a surprising but crucial function independent of potassium conductance: by organizing the F-actin cytoskeleton in mouse nerve terminals, the Kv3.3 protein facilitates slow endocytosis, rapid endocytosis, vesicle mobilization to the readily releasable pool, and recovery of synaptic depression during repetitive firing. A channel mutation that causes spinocerebellar ataxia inhibits endocytosis, vesicle mobilization, and synaptic transmission during repetitive firing by disrupting the ability of the channel to nucleate F-actin. These results unmask novel functions of potassium channels in endocytosis and vesicle mobilization crucial for sustaining synaptic transmission during repetitive firing. Potassium channel mutations that impair these "non-conducting" functions may thus contribute to generation of diverse neurological disorders.

[Design and practice of the case study methodology throughout the "Animal Genetic Principles and Breeding Methods" course].

"Animal Genetics Principles and Breeding Methods" is a main course for Master students majoring in Agriculture (Livestock) and involves a combination of theory and practice. The traditional teaching method is difficult not only to meet the requirements of modern professional degree teaching, but also for the students to master the theory and practice of genetic breeding. We have employed the case study methodology during the entire course. This paper analyzes the connotation and characteristics of the method and expounds the design and discussion of the cases. Besides, the teaching evaluation is also included. It provides a reference for the application and promotion of the case teaching method in training graduate students majoring in agriculture.

Protein Kinase C and Calmodulin Serve As Calcium Sensors for Calcium-Stimulated Endocytosis at Synapses.

Calcium influx triggers and facilitates endocytosis, which recycles vesicles and thus sustains synaptic transmission. Despite decades of studies, the underlying calcium sensor remained not well understood. Here, we examined two calcium binding proteins, protein kinase C (PKC) and calmodulin. Whether PKC is involved in endocytosis was unclear; whether calmodulin acts as a calcium sensor for endocytosis was neither clear, although calmodulin involvement in endocytosis had been suggested. We generated PKC (α or ß-isoform) and calmodulin (calmodulin 2 gene) knock-out mice of either sex and measured endocytosis with capacitance measurements, pHluorin imaging and electron microscopy. We found that these knock-outs inhibited slow (∼10-30 s) and rapid (<∼3 s) endocytosis at large calyx-type calyces, and inhibited slow endocytosis and bulk endocytosis (forming large endosome-like structures) at small conventional hippocampal synapses, suggesting the involvement of PKC and calmodulin in three most common forms of endocytosis-the slow, rapid and bulk endocytosis. Inhibition of slow endocytosis in PKC or calmodulin 2 knock-out hippocampal synapses was rescued by overexpressing wild-type PKC or calmodulin, but not calcium-binding-deficient PKC or calmodulin mutant, respectively, suggesting that calcium stimulates endocytosis by binding with its calcium sensor PKC and calmodulin. PKC and calmodulin 2 knock-out inhibited calcium-dependent vesicle mobilization to the readily releasable pool, suggesting that PKC and calmodulin may mediate calcium-dependent facilitation of vesicle mobilization. These findings shed light on the molecular signaling link among calcium, endocytosis and vesicle mobilization that are crucial in maintaining synaptic transmission and neuronal network activity.SIGNIFICANCE STATEMENT Vesicle fusion releases neurotransmitters to mediate synaptic transmission. To sustain synaptic transmission, fused vesicles must be retrieved via endocytosis. Accumulating evidence suggests that calcium influx triggers synaptic vesicle endocytosis. However, how calcium triggers endocytosis is not well understood. Using genetic tools together with capacitance measurements, optical imaging and electron microscopy, we identified two calcium sensors, including protein kinase C (α and ß isoforms) and calmodulin, for the most commonly observed forms of endocytosis: slow, rapid, and bulk. We also found that these two proteins are involved in calcium-dependent vesicle mobilization to the readily releasable pool. These results provide the molecular signaling link among calcium, endocytosis, and vesicle mobilization that are essential in sustaining synaptic transmission and neuronal network activity.

Membrane Tension Inhibits Rapid and Slow Endocytosis in Secretory Cells.

Endocytosis generates spherical or ellipsoid-like vesicles from the plasma membrane, which recycles vesicles that fuse with the plasma member during exocytosis in neurons and endocrine secretory cells. Although tension in the plasma membrane is generally considered to be an important factor in regulating endocytosis, whether membrane tension inhibits or facilitates endocytosis remains debated in the endocytosis field, and has been rarely studied for vesicular endocytosis in secretory cells. Here we report that increasing membrane tension by adjusting osmolarity inhibited both the rapid (a few seconds) and slow (tens of seconds) endocytosis in calyx-type nerve terminals containing conventional active zones and in neuroendocrine chromaffin cells. We address the mechanism of this phenomenon by computational modeling of the energy barrier that the system must overcome at the stage of membrane budding by an assembling protein coat. We show that this barrier grows with increasing tension, which may slow down or prevent membrane budding. These results suggest that in live secretory cells, membrane tension exerts inhibitory action on endocytosis.

Actin Is Crucial for All Kinetically Distinguishable Forms of Endocytosis at Synapses.

Mechanical force is needed to mediate endocytosis. Whether actin, the most abundant force-generating molecule, is essential for endocytosis is highly controversial in mammalian cells, particularly synapses, likely due to the use of actin blockers, the efficiency and specificity of which are often unclear in the studied cell. Here we addressed this issue using a knockout approach combined with measurements of membrane capacitance and fission pore conductance, imaging of vesicular protein endocytosis, and electron microscopy. We found that two actin isoforms, ß- and γ-actin, are crucial for slow, rapid, bulk, and overshoot endocytosis at large calyx-type synapses, and for slow endocytosis and bulk endocytosis at small hippocampal synapses. Polymerized actin provides mechanical force to form endocytic pits. Actin also facilitates replenishment of the readily releasable vesicle pool, likely via endocytic clearance of active zones. We conclude that polymerized actin provides mechanical force essential for all kinetically distinguishable forms of endocytosis at synapses.

α-Synuclein Mutation Inhibits Endocytosis at Mammalian Central Nerve Terminals.

α-Synuclein (α-syn) missense and multiplication mutations have been suggested to cause neurodegenerative diseases, including Parkinson's disease (PD) and dementia with Lewy bodies. Before causing the progressive neuronal loss, α-syn mutations impair exocytosis, which may contribute to eventual neurodegeneration. To understand how α-syn mutations impair exocytosis, we developed a mouse model that selectively expressed PD-related human α-syn A53T (h-α-synA53T) mutation at the calyx of Held terminals, where release mechanisms can be dissected with a patch-clamping technique. With capacitance measurement of endocytosis, we reported that h-α-synA53T, either expressed transgenically or dialyzed in the short term in calyces, inhibited two of the most common forms of endocytosis, the slow and rapid vesicle endocytosis at mammalian central synapses. The expression of h-α-synA53Tin calyces also inhibited vesicle replenishment to the readily releasable pool. These findings may help to understand how α-syn mutations impair neurotransmission before neurodegeneration. SIGNIFICANCE STATEMENT: α-Synuclein (α-syn) missense or multiplication mutations may cause neurodegenerative diseases, such as Parkinson's disease and dementia with Lewy bodies. The initial impact of α-syn mutations before neuronal loss is impairment of exocytosis, which may contribute to eventual neurodegeneration. The mechanism underlying impairment of exocytosis is poorly understood. Here we report that an α-syn mutant, the human α-syn A53T, inhibited two of the most commonly observed forms of endocytosis, slow and rapid endocytosis, at a mammalian central synapse. We also found that α-syn A53T inhibited vesicle replenishment to the readily releasable pool. These results may contribute to accounting for the widely observed early synaptic impairment caused by α-syn mutations in the progression toward neurodegeneration.

A three-pool model dissecting readily releasable pool replenishment at the calyx of held.

Although vesicle replenishment is critical in maintaining exo-endocytosis recycling, the underlying mechanisms are not well understood. Previous studies have shown that both rapid and slow endocytosis recycle into a very large recycling pool instead of within the readily releasable pool (RRP), and the time course of RRP replenishment is slowed down by more intense stimulation. This finding contradicts the calcium/calmodulin-dependence of RRP replenishment. Here we address this issue and report a three-pool model for RRP replenishment at a central synapse. Both rapid and slow endocytosis provide vesicles to a large reserve pool (RP) ~42.3 times the RRP size. When moving from the RP to the RRP, vesicles entered an intermediate pool (IP) ~2.7 times the RRP size with slow RP-IP kinetics and fast IP-RRP kinetics, which was responsible for the well-established slow and rapid components of RRP replenishment. Depletion of the IP caused the slower RRP replenishment observed after intense stimulation. These results establish, for the first time, a realistic cycling model with all parameters measured, revealing the contribution of each cycling step in synaptic transmission. The results call for modification of the current view of the vesicle recycling steps and their roles.

Brain-derived neurotrophic factor inhibits calcium channel activation, exocytosis, and endocytosis at a central nerve terminal.

Brain-derived neurotrophic factor (BDNF) is a neurotrophin that regulates synaptic function and plasticity and plays important roles in neuronal development, survival, and brain disorders. Despite such diverse and important roles, how BDNF, or more generally speaking, neurotrophins affect synapses, particularly nerve terminals, remains unclear. By measuring calcium currents and membrane capacitance during depolarization at a large mammalian central nerve terminal, the rat calyx of Held, we report for the first time that BDNF slows down calcium channel activation, including P/Q-type channels, and inhibits exocytosis induced by brief depolarization or single action potentials, inhibits slow and rapid endocytosis, and inhibits vesicle mobilization to the readily releasable pool. These presynaptic mechanisms may contribute to the important roles of BDNF in regulating synapses and neuronal circuits and suggest that regulation of presynaptic calcium channels, exocytosis, and endocytosis are potential mechanisms by which neurotrophins achieve diverse neuronal functions.

[Corrigendum] Overexpression of the chimeric plasmin-resistant VEGF165/VEGF183 (132-158) protein in murine breast cancer induces distinct vascular patterning adjacent to tumors and retarded tumor growth.

After the publication of the article, the authors noted that they had made an error regarding certain facts in their manuscript: In the abstract VEGF192 (132-158) should be changed to VEGF183 (132-158) (Page 1, Line 2). In addition, width should be changed to width2 (Page 3, Line 50). The authors regret these errors. [the original article was published in the Molecular Medicine Reports 11: 1483-1489, 2015 DOI: 10.3892/mmr.2014.2866]

Overexpression of the chimeric plasmin-resistant VEGF165/VEGF183 (132-158) protein in murine breast cancer induces distinct vascular patterning adjacent to tumors and retarded tumor growth.

A chimeric plasmin­resistant vascular endothelial growth factor (VEGF)165/VEGF183 (132-158) protein, named as VEGF183 (according to the nomenclature of VEGF), designed by a previous study, was demonstrated to have an enhanced affinity for the extracellular matrix (ECM) amongst other bioactivities. However, it is now accepted that mutant VEGFs frequently demonstrate different angiogenic activities and produce different vascular patterning from the parental molecule. The present study hypothesized that VEGF183, due to its enhanced binding affinity to the ECM, would exhibit a different angiogenic activity and produce a different vascular patterning compared to those of VEGF165. Murine breast cancer EMT­6 cells were manipulated to stably overexpress VEGF165 or VEGF183. These cells were then inoculated intradermally into BALB/c mice in order to monitor the formation of vascular patterning in skin proximal to tumors. In vivo angiogenesis experiments revealed that overexpression of VEGF183 in murine breast cancer cells resulted in irregular, disorganized and dense vascular patterning as well as induced a significant inhibition of tumor growth compared with that of VEGF165. In addition, allograft tumor immunochemical assays of VEGF183­overexpressing tumors demonstrated significantly lower vascular densities than those of VEGF165­overexpressing tumors; however, VEGF183 tumors had a significantly enlarged vascular caliber. Conversely, cell wound healing experiments revealed that VEGF183­overexpressing EMT­6 cells had significantly decreased migration rates compared with those of VEGF165­overexpressing EMT­6 cells. In conclusion, the results of the present study supported the hypothesis that the altered ECM affinity of VEGF induced structural alterations to vasculature. In addition, these results provided a novel insight into VEGF design and indirect evidence for the function of exon 8 in VEGF. [Corrected]

Calcineurin is universally involved in vesicle endocytosis at neuronal and nonneuronal secretory cells.

Calcium influx triggers and accelerates endocytosis in nerve terminals and nonneuronal secretory cells. Whether calcium/calmodulin-activated calcineurin, which dephosphorylates endocytic proteins, mediates this process is highly controversial for different cell types, developmental stages, and endocytic forms. Using three preparations that previously produced discrepant results (i.e., large calyx-type synapses, conventional cerebellar synapses, and neuroendocrine chromaffin cells containing large dense-core vesicles), we found that calcineurin gene knockout consistently slowed down endocytosis, regardless of cell type, developmental stage, or endocytic form (rapid or slow). In contrast, calcineurin and calmodulin blockers slowed down endocytosis at a relatively small calcium influx, but did not inhibit endocytosis at a large calcium influx, resulting in false-negative results. These results suggest that calcineurin is universally involved in endocytosis. They may also help explain the discrepancies among previous pharmacological studies. We therefore suggest that calcineurin should be included as a key player in mediating calcium-triggered and -accelerated vesicle endocytosis.

The yin and yang of calcium effects on synaptic vesicle endocytosis.

A large number of studies suggest that calcium triggers and accelerates vesicle endocytosis at many synapses and non-neuronal secretory cells. However, many studies show that prolonging the duration of the stimulation train, which induces more calcium influx, slows down endocytosis; and several studies suggest that instead of triggering endocytosis, calcium actually inhibits endocytosis. Here we addressed this apparent conflict at a large nerve terminal, the calyx of Held in rat brainstem, in which recent studies suggest that transient calcium increase up to tens of micromolar concentration at the micro/nano domain triggers endocytosis. By dialyzing 0-1 µM calcium into the calyx via a whole-cell pipette, we found that slow endocytosis was inhibited by calcium dialysis in a concentration-dependent manner. Thus, prolonged, small, and global calcium increase inhibits endocytosis, whereas transient and large calcium increase at the micro/nano domain triggers endocytosis and facilitates endocytosis. This yin and yang effect of calcium may reconcile apparent conflicts regarding whether calcium accelerates or inhibits endocytosis. Whether endocytosis is fast or slow depends on the net outcome between the yin and yang effect of calcium.

Identification and differential expression of microRNAs in ovaries of laying and Broody geese (Anser cygnoides) by Solexa sequencing.

BACKGROUND: Recent functional studies have demonstrated that the microRNAs (miRNAs) play critical roles in ovarian gonadal development, steroidogenesis, apoptosis, and ovulation in mammals. However, little is known about the involvement of miRNAs in the ovarian function of fowl. The goose (Anas cygnoides) is a commercially important food that is cultivated widely in China but the goose industry has been hampered by high broodiness and poor egg laying performance, which are influenced by ovarian function. METHODOLOGY/PRINCIPAL FINDINGS: In this study, the miRNA transcriptomes of ovaries from laying and broody geese were profiled using Solexa deep sequencing and bioinformatics was used to determine differential expression of the miRNAs. As a result, 11,350,396 and 9,890,887 clean reads were obtained in laying and broodiness goose, respectively, and 1,328 conserved known miRNAs and 22 novel potential miRNA candidates were identified. A total of 353 conserved microRNAs were significantly differentially expressed between laying and broody ovaries. Compared with miRNA expression in the laying ovary, 127 miRNAs were up-regulated and 126 miRNAs were down-regulated in the ovary of broody birds. A subset of the differentially expressed miRNAs (G-miR-320, G-miR-202, G-miR-146, and G-miR-143*) were validated using real-time quantitative PCR. In addition, 130,458 annotated mRNA transcripts were identified as putative target genes. Gene ontology annotation and KEGG (Kyoto Encyclopedia of Genes and Genomes) pathway analysis suggested that the differentially expressed miRNAs are involved in ovarian function, including hormone secretion, reproduction processes and so on. CONCLUSIONS: The present study provides the first global miRNA transcriptome data in A. cygnoides and identifies novel and known miRNAs that are differentially expressed between the ovaries of laying and broody geese. These findings contribute to our understanding of the functional involvement of miRNAs in the broody period of goose.

Most vesicles in a central nerve terminal participate in recycling.

Studies over the last decade using FM dyes to label vesicles at many terminals, including the calyx-type nerve terminal, led to a well accepted "principle" that only a small fraction of vesicles (∼5-20%) participate in recycling under physiological conditions. This principle imposes a large challenge in maintaining synaptic transmission during repetitive firing, because the small recycling pool may limit the number of available vesicles for release and nerve terminals would have to distinguish the recycling pool from the reserve pool and keep reserve pool vesicles from being used. By recording the presynaptic capacitance changes and the postsynaptic EPSC at rat calyx of Held synapses in the absence or presence of transmitter glutamate in nerve terminals, we developed a new method to count functional recycling vesicles. We found that essentially all vesicles in calyces participated in recycling, challenging the small-recycling-pool principle established by FM dye labeling. Nerve terminals may use all available vesicles to maximize their ability in maintaining synaptic transmission during repetitive firing.

SNARE proteins synaptobrevin, SNAP-25, and syntaxin are involved in rapid and slow endocytosis at synapses.

Rapid endocytosis, which takes only a few seconds, is widely observed in secretory cells. Although it is more efficient in recycling vesicles than in slow clathrin-mediated endocytosis, its underlying mechanism, thought to be clathrin independent, is largely unclear. Here, we report that cleavage of three SNARE proteins essential for exocytosis, including synaptobrevin, SNAP-25, and syntaxin, inhibited rapid endocytosis at the calyx of Held nerve terminal, suggesting the involvement of the three SNARE proteins in rapid endocytosis. These SNARE proteins were also involved in slow endocytosis. In addition, SNAP-25 and syntaxin facilitated vesicle mobilization to the readily releasable pool, most likely via their roles in endocytosis and/or exocytosis. We conclude that both rapid and slow endocytosis share the involvement of SNARE proteins. The dual role of three SNARE proteins in exo- and endocytosis suggests that SNARE proteins may be molecular substrates contributing to the exocytosis-endocytosis coupling, which maintains exocytosis in secretory cells.

Voltage-dependent calcium channels at the plasma membrane, but not vesicular channels, couple exocytosis to endocytosis.

Although calcium influx triggers endocytosis at many synapses and non-neuronal secretory cells, the identity of the calcium channel is unclear. The plasma membrane voltage-dependent calcium channel (VDCC) is a candidate, and it was recently proposed that exocytosis transiently inserts vesicular calcium channels at the plasma membrane, thus triggering endocytosis and coupling it to exocytosis, a mechanism suggested to be conserved from sea urchin to human. Here, we report that the vesicular membrane, when inserted into the plasma membrane upon exocytosis, does not generate a calcium current or calcium increase at a mammalian nerve terminal. Instead, VDCCs at the plasma membrane, including the P/Q-type, provide the calcium influx to trigger rapid and slow endocytosis and, thus, couple endocytosis to exocytosis. These findings call for reconsideration of the vesicular calcium channel hypothesis. They are likely to apply to many synapses and non-neuronal cells in which VDCCs control exocytosis, and exocytosis is coupled to endocytosis.