Yuechueng Liu

Reliable and Global Measurement of Fluorescence Resonance Energy Transfer Using Fluorescence Microscopes

Green fluorescence protein (GFP)-based fluorescence resonance energy transfer (FRET) is increasingly used in investigation of inter- and intramolecular interactions in living cells. In this report, we present a modified method for FRET quantification in cultured cells using conventional fluorescence microscopy. To reliably measure FRET, three positive control constructs in which a cyan fluorescence protein and a yellow fluorescence protein were linked by peptides of 15, 24, or 37 amino acid residues were prepared. FRET was detected using a spectrofluorometer, a laser scanning confocal microscope, and an inverted fluorescence microscope. Three calculation methods for FRET quantification using fluorescence microscopes were compared. By normalization against expression levels of GFP fusion proteins, the modified method gave consistent FRET values that could be compared among different cells with varying protein expression levels. Whole-cell global analysis using this method allowed FRET measurement with high spatial resolutions. Using such a procedure, the interaction of synaptic proteins syntaxin and the synaptosomal associated protein of 25 kDa (SNAP-25) was examined in PC12 cells, which showed strong FRET on plasma membranes. These results demonstrate the effectiveness of the modified method for FRET measurement in live cell systems.

Characterization of the palmitoylation domain of SNAP-25.

Abstract: SNAP‐25 (synaptosomal associated protein of 25 kDa) is a neural specific protein that has been implicated in the synaptic vesicle docking and fusion process. It is tightly associated with membranes, and it is one of the major palmitoylated proteins found in neurons. The functional role of palmitoylation for SNAP‐25 is unclear. In this report, we show that the palmitate of SNAP‐25 is rapidly turned over in PC12 cells, with a half‐life of ∼3 h, and the half‐life for the protein is 8 h. Mutation of Cys to Ser at positions 85, 88, 90, and 92 reduced the palmitoylation to 9, 21, 42, and 35% of the wild‐type protein, respectively. Additional mutations of either Cys85,88 or Cys90,92 nearly abolished palmitoylation of the protein. A similar effect on membrane binding for the mutant SNAP‐25 was observed, which correlated with the degree of palmitoylation. These results suggest that all four Cys residues are involved in palmitoylation and that membrane association of SNAP‐25 may be regulated through dynamic palmitoylation.

Differential roles of ERK and JNK in early and late stages of neuritogenesis: a study in a novel PC12 model system

The rat pheochromocytoma PC12 cell line has been an invaluable model system for studying neuritogenesis. Nerve growth factor (NGF) elicits multiple aspects of neurite outgrowth in PC12 cells. It is therefore difficult to dissect and assign an individual signaling pathway to each stage of neuritogenesis. We have recently reported the isolation of a variant PC12 cell line, PC12‐N1 (N1), which spontaneously extends neuritic processes and exhibits an increased sensitivity to NGF. Here, we show that, under different culture conditions, the cells display three distinct phases of neuritogenesis consisting of neurite initiation, rapid neurite elongation, and a maturation process characterized by the thickening of neurites and increase in cell soma sizes. We demonstrate that signaling through ERK, but not p38 or JNK, is required for the spontaneous neurite initiation and extension. Treatment with low concentrations of NGF induces rapid neurite elongation without affecting neurite branching and cell soma sizes. Such a rapid neurite outgrowth can be blocked by the inhibition of ERK, but not JNK, activities. In the presence of higher concentrations of NGF, the N1 cells undergo further differentiation with many characteristics of mature neurons in culture, e.g. larger cell soma and numerous branches/connections. This process can be completely blocked by inhibiting ERK or JNK activities using specific inhibitors. These results suggest that ERK and JNK signals play different roles in neuritogenesis, and that JNK activity is essential in the late stages of neuritogenesis. Furthermore, our results demonstrate that signaling dosage is important in the activation of a specific pathway, leading to distinctive biological outcomes.

Participation of syntaxin 1A in membrane trafficking involving neurite elongation and membrane expansion.

Syntaxin 1A has been implicated to play an important role in neurotransmitter release by regulating synaptic vesicle fusion. The protein is also suggested to be required for other types of membrane fusion such as cellularization during embryonic development. In the current study, we overexpressed syntaxin 1A, SNAP‐25b, and VAMP‐2 in PC12 cells using recombinant adenoviruses, and determined their effects on membrane trafficking involving neurite outgrowth. It was found that overexpression of syntaxin 1A inhibited NGF‐induced neurite extension, and the expressed syntaxin was localized to the plasma membrane, intracellular membranes, and the neurite tips. SNAP‐25 overexpression slightly enhanced neurite elongation, whereas no significant changes in neurite growth was observed in VAMP‐overexpressing cells. The effect of syntaxin 1A in general membrane trafficking was further studied by transient transfection of non‐neuronal cells. Syntaxin 1A expression in HEK 239 and NIH3T3‐L1 caused the cells to lose their normal morphology, leading to round and smaller cells. Deletion of the C‐terminal sequence containing the H3 helix and the membrane anchoring domains of syntaxin abolished its ability to induce cell morphology changes, whereas removal of the N‐terminal 1‐170 amino acid sequence did not affect this activity. These findings suggest that in addition to its well documented role in synaptic vesicle fusion, syntaxin may function in other non‐synaptic membrane trafficking such as neurite outgrowth and membrane expansion. J. Neurosci. Res. 61:321–328, 2000.

Localization and Function of SolubleN-Ethylmaleimide-sensitive Factor Attachment Protein-25 and Vesicle-associated Membrane Protein-2 in Functioning Gastric Parietal Cells

The solubleN-ethylmaleimide-sensitive factor attachment protein of 25 kDa (SNAP-25) plays an important role in vesicle trafficking. Together with vesicle-associated membrane protein-2 (VAMP-2) and syntaxin, SNAP-25 forms a ternary complex implicated in docking and fusion of secretory vesicles with the plasma membrane during exocytosis. These so-called SNARE proteins are believed to regulate tubulovesicle trafficking and fusion during the secretory cycle of the gastric parietal cell. Here we examined the cellular localization and functional importance of SNAP-25 in parietal cell cultures. Adenoviral constructs were used to express SNAP-25 tagged with cyan fluorescent protein, VAMP-2 tagged with yellow fluorescent protein, and SNAP-25 in which the C-terminal 25 amino acids were deleted (SNAP-25 Δ181–206). Membrane fractionation experiments and fluorescent imaging showed that SNAP-25 is localized to the apical plasma membrane. The expression of the mutant SNAP-25 Δ181–226 inhibited the acid secretory response of parietal cells. Also, SNAP Δ181–226 bound poorly in vitro with recombinant syntaxin-1 compared with wild type SNAP-25, indicating that pairing between syntaxin-1 and SNAP-25 is required for parietal cell activation. Dual expression of SNAP-25 tagged with cyan fluorescent protein and VAMP-2 tagged with yellow fluorescent protein revealed a dynamic change in distribution associated with acid secretion. In resting cells, SNAP-25 is at the apical plasma membrane and VAMP-2 is associated with cytoplasmic H,K-ATPase-rich tubulovesicles. After stimulation, the two proteins co-localize on the apical plasma membrane. These data demonstrate the functional significance of SNAP-25 as a SNARE protein in the parietal cell and show the dynamic stimulation-associated redistribution of VAMP-2 from H,K-ATPase-rich tubulovesicles to co-localize with SNAP-25 on the apical plasma membrane.

Differential turnover of syntaxin and SNAP-25 during synaptogenesis in cultured cerebellar granule neurons

In order to investigate the molecular mechanism underlying synaptogenesis, we examined the dynamics and stability of syntaxin 1A and SNAP‐25 in cultured cerebellar granule cells. In neurons cultured for less than 5 days in vitro (DIV), syntaxin was highly expressed with a half‐life of >48 hours. SNAP‐25 was also expressed at 5 DIV, but at a lower level and with a much shorter half‐life of 16 hours. As the neurons matured and established synpatic connections, the expression of both proteins increased steadily, with the more rapid increase between 5 DIV and 8 DIV associated with SNAP‐25. The half‐life of syntaxin was slightly increased in the mature neurons. SNAP‐25, however, showed an increased half‐life of about 35 hours. These results suggested that the dynamics and stability of the t‐SNAREs are differentially modulated during synaptogenesis, which may be important in establishing and maintaining synaptic connections. J. Neurosci. Res. 53:670–676, 1998.

Functional role of JNK in neuritogenesis of PC12-N1 cells

JNKs, also known as SAPKs, are activated in response to a wide variety of factors including growth factors, cytokines, UV radiation, and heat shock. In the rat pheochromocytoma PC12 cells, the JNK signaling pathway mediates diverse functions such as differentiation and apoptosis. We have previously shown that activated JNK is required for later stages of neuritogeneis induced by NGF in a variant PC12 cell line (N1). Here, the functional role of JNK in N1 cells is further investigated. We show that NGF treatment, which induces extensive neurite branching and cell soma enlargement in the N1 cells, stimulates a biphasic activation of JNK. The first phase of activation is rapid and transient, beginning at 15 min after NGF exposure and lasting approximately 45 min. The second phase of activation is sustained, beginning at 9-12 h of NGF treatment and lasting for at least 24 h. Similar biphasic pattern of JNK activation is also observed in the parental PC12 cells. Using the specific JNK inhibitor SP600125, we show that the biphasic activation is necessary for neurite outgrowth and branching, and that inhibition of either phase suppresses neuritogenesis in the N1 cells. These results suggest that dynamic JNK activation may play a key role in neurite outgrowth during neuronal development.

An immunohistochemical method that distinguishes free from complexed SNAP‐25

Soluble N‐ethylmaleimide‐sensitive fusion protein (NSF) attachment protein receptor (SNARE) complexes composed of target (t‐) SNAREs syntaxin and SNAP‐25 and vesicle SNARE synaptobrevin play an essential role in neurosecretion. It is hypothesized that a transient intermediate complex between the t‐SNAREs is formed during the assembly of the ternary complex. The existence of the t‐SNARE binary complexes in vivo, however, has not been demonstrated. By using an affinity absorption scheme with preformed syntaxin–SNAP‐25 complexes, we isolated antibodies capable of distinguishing free SNAP‐25 from those associated with syntaxin. By semiquantitative immunohistochemistry, we estimated that, in cultured cerebellar neurons, the majority of SNAP‐25 existed as complexes. Compared with the cultured neurons, PC12 cells expressed significantly less syntaxin, and we found that SNAP‐25 was primarily in free forms. In contrast, a PC12 line that stably expressed a recombinant syntaxin showed a marked increase in SNAP‐25 complexes. By using fluorescence resonance energy transfer (FRET) techniques, we observed FRET between cyan fluorescence protein–syntaxin and yellow fluorescence protein–SNAP‐25 fusion proteins expressed in COS‐7 and PC12 cells, suggesting a physiological interaction between syntaxin and SNAP‐25. Our results demonstrate that, unlike what was previously hypothesized, syntaxin and SNAP‐25 exist preferably as stable binary complexes in neurons. These findings offer novel insight into the mechanisms underlying the initiation and regulation of SNARE complex assembly.

SNAP-25 Functional Domains in SNARE Core Complex Assembly and Glutamate Release of Cerebellar Granule Cells

Synaptosomal associated protein of 25 kDa (SNAP-25) is a member of the SNARE protein complex that has been implicated in synaptic vesicle docking and fusion. In this report, we have generated SNAP-25 mutants and assayed their functions in SNARE complex formation and glutamate release from cultured rat cerebellar granule cells. In vitro binding studies show that a deletion mutant lacking the C-terminal 181–206 amino acid sequence inhibits the formation of the SNARE core complex. Additional deletion of an N-terminal 1–31 amino acid sequence abolished this inhibitory activity. Adenovirus-mediated gene transfer is used to overexpress wild-type and mutant SNAP-25 in cerebellar granule cells. Neurons overexpressing the wild-type protein show slight reductions in glutamate release, ranging from 10 to 15% in both the developing and mature granule cells. A 30–35% inhibition is obtained with the C-terminal deletion mutant, and the inhibitory effect is abolished in the N- and C-terminal double deletion mutant. These results demonstrate that the SNARE core complex exists in a dynamic and reversible state, and the formation of the core complex is necessary for neurotransmitter release in neurons.

Novel method for the labeling of distant neuromuscular junctions

Essential to understanding the roles proteins and structural elements play at the synapse is to understand the development, remodeling and reinnervation of peripheral neuromuscular junctions. It has, however, been a challenging task to label and visualize neuromuscular junctions. In this paper we demonstrate how adenovirus technology can be combined with intraspinal microinjection techniques to follow both the development and the reinnervation of a distant peripheral neuromuscular junction in the rat. A recombinant adenovirus containing VAMP‐2 (synaptobrevin‐2) was fused to the green fluorescent protein (GFP) and microinjected into the region of the lumbar motor neurons. We were able to follow the neuronal incorporation, axonal transport and synaptic localization of the GFP‐VAMP‐2 using fluorescence microscopy. GFP‐VAMP‐2 was found in neuronal cell bodies, selected sciatic nerve axons and was concentrated in the presynaptic nerve terminal. During reinnervation of the neuromuscular junction, GFP‐VAMP‐2 allows us to follow the time course of junctional reinnervation. Thus, the microinjection of microliter amounts of labeled recombinant virus into locations far distant from target regions can be used to efficiently study the formation of neuromuscular junctions with a minimum of trauma to the animal. J. Neurosci. Res. 61:61–66, 2000.