Biomedical subjects

Weiping Han

Publications and source records attributed to Weiping Han.

11 recordsLinked to original sources

PubMed2006

Redundant functions of RIM1alpha and RIM2alpha in Ca(2+)-triggered neurotransmitter release.

Alpha-RIMs (RIM1alpha and RIM2alpha) are multidomain active zone proteins of presynaptic terminals. Alpha-RIMs bind to Rab3 on synaptic vesicles and to Munc13 on the active zone via their N-terminal region, and interact with other synaptic proteins via their central and C-terminal regions. Although RIM1alpha has been well characterized, nothing is known about the function of RIM2alpha. We now show that RIM1alpha and RIM2alpha are expressed in overlapping but distinct patterns throughout the brain. To examine and compare their functions, we generated knockout mice lacking RIM2alpha, and crossed them with previously produced RIM1alpha knockout mice. We found that deletion of either RIM1alpha or RIM2alpha is not lethal, but ablation of both alpha-RIMs causes postnatal death. This lethality is not due to a loss of synapse structure or a developmental change, but to a defect in neurotransmitter release. Synapses without alpha-RIMs still contain active zones and release neurotransmitters, but are unable to mediate normal Ca(2+)-triggered release. Our data thus demonstrate that alpha-RIMs are not essential for synapse formation or synaptic exocytosis, but are required for normal Ca(2+)-triggering of exocytosis.

Action Potentials↗

PubMed2006

Synapsins regulate use-dependent synaptic plasticity in the calyx of Held by a Ca2+/calmodulin-dependent pathway.

Synapsins are abundant synaptic-vesicle phosphoproteins that are known to regulate neurotransmitter release but whose precise function has been difficult to pinpoint. Here, we use knockout mice to analyze the role of synapsins 1 and 2 in the calyx of Held synapse, allowing precise measurements of neurotransmitter release. We find that deletion of synapsins did not induce significant changes in spontaneous release or release evoked by isolated action potentials (APs) and did not alter the size of the readily releasable vesicle pool (RRP), the kinetics of RRP depletion, or the rate of recovery of the RRP after depletion. Deletion of synapsins, however, did increase use-dependent synaptic depression induced by a high-frequency stimulus train (> or = 50 Hz). The increased depression was due to a decrease in the fraction of the RRP, whose release was evoked by APs late in the stimulus train. The effect of synapsin deletions was occluded by intracellular application of the Ca2+-chelator EGTA or of a calmodulin inhibitor. Our results show that synapsins boost the release probability during high-frequency stimulation and suggest that this effect involves Ca2+/calmodulin-dependent phosphorylation of synapsins.

Action Potentials↗

PubMed2004

C-terminal ECFP fusion impairs synaptotagmin 1 function: crowding out synaptotagmin 1.

To allow the monitoring of synaptotagmin 1 trafficking in vivo, we generated transgenic mice expressing a synaptotagmin 1-enhanced cyan fluorescent protein (ECFP) fusion protein under control of the Thy1 promoter. Transgenic synaptotagmin 1-ECFP is expressed throughout the brain where it localizes to synapses and marks synapses in vivo. However, when we crossed transgenic synaptotagmin 1-ECFP mice with synaptotagmin 1 knock-out mice, we detected no rescue of survival or function. Furthermore, viral overexpression of synaptotagmin 1-ECFP in synaptotagmin 1-deficient neurons failed to restore normal Ca2+-triggered release, whereas overexpression of wild type synaptotagmin 1 did so efficiently. To determine whether synaptotagmin 1-ECFP is non-functional because the ECFP-fusion interferes with its biochemical activities, we measured Ca2+-independent binding of synaptotagmin 1-ECFP to SNARE complexes, and Ca2+-dependent binding of synaptotagmin 1-ECFP to phospholipids and to itself. Although the apparent Ca2+ affinity of synaptotagmin 1-ECFP was decreased compared with wild type synaptotagmin 1, we observed no major changes in Ca2+-dependent or -independent activities, indicating that the non-functionality of the synaptotagmin 1-ECFP fusion protein was not because of inactivation of its biochemical properties. These data suggest that synaptotagmin 1-ECFP is suitable for monitoring synaptic vesicle traffic in vivo because the synaptotagmin 1-ECFP marks synaptic vesicles without participating in exocytosis. In addition, the data demonstrate that synaptotagmin 1 function requires a free C terminus, possibly because of spatial constraints at the release sites.

Animals↗

PubMed2004

Evolutionarily conserved multiple C2 domain proteins with two transmembrane regions (MCTPs) and unusual Ca2+ binding properties.

C2 domains are primarily found in signal transduction proteins such as protein kinase C, which generally contain a single C2 domain, and in membrane trafficking proteins such as synaptotagmins, which generally contain multiple C2 domains. In both classes of proteins, C2 domains usually regulate the respective protein's function by forming Ca(2+)-dependent or Ca(2+)-independent phospholipid complexes. We now describe MCTPs (multiple C2 domain and transmembrane region proteins), a novel family of evolutionarily conserved C2 domain proteins with unusual Ca(2+)-dependent properties. MCTPs are composed of a variable N-terminal sequence, three C2 domains, two transmembrane regions, and a short C-terminal sequence. The invertebrate organisms Caenorhabditis elegans and Drosophila melanogaster express a single MCTP gene, whereas vertebrates express two MCTP genes (MCTP1 and MCTP2) whose primary transcripts are extensively alternatively spliced. Most of the MCTP sequences, in particular the C2 domains, are highly conserved. All MCTP C2 domains except for the second C2 domain of MCTP2 include a perfect Ca2+/phospholipid-binding consensus sequence. To determine whether the C2 domains of MCTPs actually function as Ca2+/phospholipid-binding modules, we analyzed their Ca2+ and phospholipid binding properties. Surprisingly, we found that none of the three MCTP1 C2 domains interacted with negatively charged or neutral phospholipids in the presence or absence of Ca2+. However, Ca2+ titrations monitored via intrinsic tryptophan fluorescence revealed that all three C2 domains bound Ca2+ in the absence of phospholipids with a high apparent affinity (EC50 of approximately 1.3-2.3 microM). Our data thus reveal that MCTPs are evolutionarily conserved C2 domain proteins that are unusual in that the C2 domains are anchored in the membrane by two closely spaced transmembrane regions and represent Ca(2+)-binding but not phospholipid-binding modules.

Amino Acid Sequence↗

PubMed2004

N-glycosylation is essential for vesicular targeting of synaptotagmin 1.

Synaptotagmins 1 and 7 are candidate Ca(2+) sensors for exocytosis localized to synaptic vesicles and plasma membranes, respectively. We now show that the N-terminal intraluminal sequence of synaptotagmin 1, when transplanted onto synaptotagmin 7, redirects synaptotagmin 7 from the plasma membrane to secretory vesicles. Conversely, mutation of the N-terminal N-glycosylation site of synaptotagmin 1 redirects synaptotagmin 1 from vesicles to the plasma membrane. In cultured hippocampal neurons, the plasma membrane-localized mutant of synaptotagmin 1 suppressed the readily releasable pool of synaptic vesicles, whereas wild-type synaptotagmin 1 did not. In addition to the intraluminal N-glycosylation site, the cytoplasmic C(2) domains of synaptotagmin 1 were required for correct targeting but could be functionally replaced by the C(2) domains of synaptotagmin 7. Our data suggest that the intravesicular N-glycosylation site of synaptotagmin 1 collaborates with its cytoplasmic C(2) domains in directing synaptotagmin 1 to synaptic vesicles via a novel N-glycosylation-dependent mechanism.

Aging↗

PubMed2003

A conserved GXXXG motif in APH-1 is critical for assembly and activity of the gamma-secretase complex.

The multipass membrane protein APH-1, found in the gamma-secretase complex together with presenilin, nicastrin, and PEN-2, is essential for Notch signaling in Caenorhabditis elegans embryos and is required for intramembrane proteolysis of Notch and beta-amyloid precursor protein in mammalian and Drosophila cells. In C. elegans, a mutation of the conserved transmembrane Gly123 in APH-1 (mutant or28) leads to a notch/glp-1 loss-of-function phenotype. In this study, we show that the corresponding mutation in mammalian APH-1aL (G122D) disrupts the physical interaction of APH-1aL with hypoglycosylated immature nicastrin and the presenilin holoprotein as well as with mature nicastrin, presenilin, and PEN-2. The G122D mutation also reduced gamma-secretase activity in intramembrane proteolysis of membrane-tethered Notch. Moreover, we found that the conserved transmembrane Gly122, Gly126, and Gly130 in the fourth transmembrane region of mammalian APH-1aL are part of the membrane helix-helix interaction GXXXG motif and are essential for the stable association of APH-1aL with presenilin, nicastrin, and PEN-2. These findings suggest that APH-1 plays a GXXXG-dependent scaffolding role in both the initial assembly and subsequent maturation and maintenance of the active gamma-secretase complex.

Amino Acid Motifs↗

PubMed2003

Synaptotagmin 7 splice variants differentially regulate synaptic vesicle recycling.

The speed of synaptic vesicle recycling determines the efficacy of neurotransmission during repetitive stimulation. Synaptotagmins are synaptic C(2)-domain proteins that are involved in exocytosis, but have also been linked to endocytosis. We now demonstrate that upon expression in transfected neurons, a short splice variant of synaptotagmin 7 that lacks C(2)-domains accelerates endocytic recycling of synaptic vesicles, whereas a longer splice variant that contains C(2)-domains decelerates recycling. These results suggest that alternative splicing of synaptotagmin 7 acts as a molecular switch, which targets vesicles to fast and slow recycling pathways.

Alternative Splicing↗

PubMed2003

Unexpected mobility variation among individual secretory vesicles produces an apparent refractory neuropeptide pool.

Most stored neuropeptide cannot be released from nerve terminals suggesting the existence of a refractory pool of dense core vesicles (DCVs). Past fluorescence photobleaching recovery, single particle tracking and release experiments suggested that the refractory neuropeptide pool corresponds to a distinct immobile fraction of cytoplasmic DCVs. However, tracking of hundreds of individual green fluorescent protein-labeled neuropeptidergic vesicles by wide-field or evanescent-wave microscopy shows that a separate immobile fraction is not evident. Instead, the DCV diffusion coefficient (D) distribution is unusually broad and asymmetric. Furthermore, the distribution shifts with a release facilitator. This unexpected variation, which could reflect heterogeneity among vesicles or in their medium, is shown to generate the appearance of a regulated refractory neuropeptide pool.

Animals↗

PubMed2002

Mammalian APH-1 interacts with presenilin and nicastrin and is required for intramembrane proteolysis of amyloid-beta precursor protein and Notch.

Presenilin and nicastrin are essential components of the gamma-secretase complex that is required for the intramembrane proteolysis of an increasing number of membrane proteins including the amyloid-beta precursor protein (APP) and Notch. By using co-immunoprecipitation and nickel affinity pull-down approaches, we now show that mammalian APH-1 (mAPH-1), a conserved multipass membrane protein, physically associates with nicastrin and the heterodimers of the presenilin amino- and carboxyl-terminal fragments in human cell lines and in rat brain. Similar to the loss of presenilin or nicastrin, the inactivation of endogenous mAPH-1 using small interfering RNAs results in the decrease of presenilin levels, accumulation of gamma-secretase substrates (APP carboxyl-terminal fragments), and reduction of gamma-secretase products (amyloid-beta peptides and the intracellular domains of APP and Notch). These data indicate that mAPH-1 is probably a functional component of the gamma-secretase complex required for the intramembrane proteolysis of APP and Notch.

Amino Acid Sequence↗

PubMed2002

Synaptotagmins form a hierarchy of exocytotic Ca(2+) sensors with distinct Ca(2+) affinities.

Synaptotagmins constitute a large family of membrane proteins implicated in Ca(2+)-triggered exocytosis. Structurally similar synaptotagmins are differentially localized either to secretory vesicles or to plasma membranes, suggesting distinct functions. Using measurements of the Ca(2+) affinities of synaptotagmin C2-domains in a complex with phospholipids, we now show that different synaptotagmins exhibit distinct Ca(2+) affinities, with plasma membrane synaptotagmins binding Ca(2+) with a 5- to 10-fold higher affinity than vesicular synaptotagmins. To test whether these differences in Ca(2+) affinities are functionally important, we examined the effects of synaptotagmin C2-domains on Ca(2+)-triggered exocytosis in permeabilized PC12 cells. A precise correlation was observed between the apparent Ca(2+) affinities of synaptotagmins in the presence of phospholipids and their action in PC12 cell exocytosis. This was extended to PC12 cell exocytosis triggered by Sr(2+), which was also selectively affected by high-affinity C2-domains of synaptotagmins. Together, our results suggest that Ca(2+) triggering of exocytosis involves tandem Ca(2+) sensors provided by distinct plasma membrane and vesicular synaptotagmins. According to this hypothesis, plasma membrane synaptotagmins represent high-affinity Ca(2+) sensors involved in slow Ca(2+)-dependent exocytosis, whereas vesicular synaptotagmins function as low-affinity Ca(2+) sensors specialized for fast Ca(2+)-dependent exocytosis.

Animals↗

PubMed2002

A new green fluorescent protein construct for localizing and quantifying peptide release.

Green fluorescent protein (GFP)-tagged secretory proteins recently have been used for studying packaging of peptides, secretory vesicle dynamics, and regulation of peptide release. In cells in which release occurs from sites with an abundance of vesicles, it is difficult to resolve the exact location of individual exocytotic events with standard wide-field epifluorescence microscopy. Furthermore, current GFP constructs are not well suited for real-time measurement of peptide release from large numbers of cells. Here, we describe a new pH-sensitive construct that is designed for localizing and quantifying release of neuropeptides and peptide hormones. Specifically, the yellow GFP variant called Topaz was fused to proAtrial natriuretic peptide (proANP, also called proAtrial natriuretic factor). The fluorescence of this fusion protein is low in normally acidic secretory vesicles but increases approximately 10-fold upon neutralization. Furthermore, it is released upon depolarization of PC12 cells. Finally, individual release events can be detected as brief localized flashes of fluorescence. ProANF-Tpz should prove useful for studying single release events by wide-field epifluorescence microscopy and for fluorometer-based real-time peptide release measurements from high numbers of cells.

Animals↗