Synaptotagmin-1 is a bidirectional Ca2+ sensor for neuronal endocytosis.

Exocytosis and endocytosis are tightly coupled. In addition to initiating exocytosis, Ca2+ plays critical roles in exocytosis­endocytosis coupling in neurons and nonneuronal cells. Both positive and negative roles of Ca2+ in endocytosis have been reported; however, Ca2+ inhibition in endocytosis remains debatable with unknown mechanisms. Here, we show that synaptotagmin-1 (Syt1), the primary Ca2+ sensor initiating exocytosis, plays bidirectional and opposite roles in exocytosis­endocytosis coupling by promoting slow, small-sized clathrin-mediated endocytosis but inhibiting fast, large-sized bulk endocytosis. Ca2+-binding ability is required for Syt1 to regulate both types of endocytic pathways, the disruption of which leads to inefficient vesicle recycling under mild stimulation and excessive membrane retrieval following intense stimulation. Ca2+-dependent membrane tubulation may explain the opposite endocytic roles of Syt1 and provides a general membrane-remodeling working model for endocytosis determination. Thus, Syt1 is a primary bidirectional Ca2+ sensor facilitating clathrin-mediated endocytosis but clamping bulk endocytosis, probably by manipulating membrane curvature to ensure both efficient and precise coupling of endocytosis to exocytosis.

Interacting proteins of the essential two-component system YycFG in Bacillus subtilis.

Two-component signal transduction systems (TCSs) play a major role in adaption and survival of microorganisms in a dynamic and sometimes dangerous environment. YycFG is an essential TCS for many Gram-positive bacteria, such as Bacillus subtilis, which regulates many important biological processes. However, its functional essentiality remains largely unknown. Here, we report several YycFG interacting proteins through coimmunoprecipitation (Co-IP) and mass spectrometry (MS) analyses. We engineered the B. subtilis genome by a knock-in approach to express YycG with a C-terminal Flag and YycF with an N-terminal HA tag. Immunoprecipitated fractions using anti-Flag or anti-HA agarose were subjected to MS analyses. A total of 41 YycG interacting proteins and four YycF interacting proteins were identified, most of which are involved in cellular metabolic processes, including cell wall synthesis and modification. The interactions of YycG with AsnB and FabL, as examples, were further validated in vitro. This study provided a clue that YycFG may be directly involved in regulation of bacterial central metabolic pathways.

STED Imaging of Vesicular Endocytosis in the Synapse.

Endocytosis is a fundamental biological process that couples exocytosis to maintain the homeostasis of the plasma membrane and sustained neurotransmission. Super-resolution microscopy enables optical imaging of exocytosis and endocytosis in live cells and makes an essential contribution to understanding molecular mechanisms of endocytosis in neuronal somata and other types of cells. However, visualization of exo-endocytic events at the single vesicular level in a synapse with optical imaging remains a great challenge to reveal mechanisms governing the synaptic exo-endocytotic coupling. In this protocol, we describe the technical details of stimulated emission depletion (STED) imaging of synaptic endocytosis at the single-vesicle level, from sample preparation and microscopy calibration to data acquisition and analysis.

An ACC-VTA-ACC positive-feedback loop mediates the persistence of neuropathic pain and emotional consequences.

The central mechanisms underlying pain chronicity remain elusive. Here, we identify a reciprocal neuronal circuit in mice between the anterior cingulate cortex (ACC) and the ventral tegmental area (VTA) that mediates mutual exacerbation between hyperalgesia and allodynia and their emotional consequences and, thereby, the chronicity of neuropathic pain. ACC glutamatergic neurons (ACCGlu) projecting to the VTA indirectly inhibit dopaminergic neurons (VTADA) by activating local GABAergic interneurons (VTAGABA), and this effect is reinforced after nerve injury. VTADA neurons in turn project to the ACC and synapse to the initial ACCGlu neurons to convey feedback information from emotional changes. Thus, an ACCGlu-VTAGABA-VTADA-ACCGlu positive-feedback loop mediates the progression to and maintenance of persistent pain and comorbid anxiodepressive-like behavior. Disruption of this feedback loop relieves hyperalgesia and anxiodepressive-like behavior in a mouse model of neuropathic pain, both acutely and in the long term.

Synaptotagmins: Beyond Presynaptic Neurotransmitter Release.

Synaptotagmins (Syts) are well-established primary Ca2+ sensors to initiate presynaptic neurotransmitter release. They also play critical roles in the docking, priming, and fusion steps of exocytosis, as well as the tightly coupled exo-endocytosis, in presynapses. A recent study by Awasthi and others (2019) shows that Syt3 Ca2+-dependently modulates the postsynaptic receptor endocytosis and thereby promotes the long-term depression (LTD) and the decay of long-term potentiation (LTP). This work highlights the importance of Syt3 in modulating long-term synaptic plasticity and, importantly, extends the function of Syt proteins from presynaptic neurotransmitter release to a new promising postsynaptic receptor internalization.