CiteULike: lechristophe Cheng

A Presynaptic Giant Ankyrin Stabilizes the NMJ through Regulation of Presynaptic Microtubules and Transsynaptic Cell Adhesion

Jan Pielage — 2008-04-24T14:22:25-00:00

Neuron, Vol. 58, No. 2. (24 April 2008), pp. 195-209.

Summary In a forward genetic screen for mutations that destabilize the neuromuscular junction, we identified a novel long isoform of Drosophila ankyrin2 (ank2-L). We demonstrate that loss of presynaptic Ank2-L not only causes synapse disassembly and retraction but also disrupts neuronal excitability and NMJ morphology. We provide genetic evidence that ank2-L is necessary to generate the membrane constrictions that normally separate individual synaptic boutons and is necessary to achieve the normal spacing of subsynaptic protein domains, including the normal organization of synaptic cell adhesion molecules. Mechanistically, synapse organization is correlated with a lattice-like organization of Ank2-L, visualized using extended high-resolution structured-illumination microscopy. The stabilizing functions of Ank2-L can be mapped to the extended C-terminal domain that we demonstrate can directly bind and organize synaptic microtubules. We propose that a presynaptic Ank2-L lattice links synaptic membrane proteins and spectrin to the underlying microtubule cytoskeleton to organize and stabilize the presynaptic terminal.

Cholesterol Level Regulates Endosome Mobility via Rab Proteins.

Hongtao Chen — 2008-01-03T10:38:14-00:00

Biophys J (2 November 2007)

The role of cholesterol in regulation of endosome motility was investigated by monitoring the intracellular trafficking of endocytosed folate receptors (FRs) labeled with fluorescent folate conjugates. Real-time fluorescence imaging of HeLa cells transfected with GFP-tubulin revealed that FR-containing endosomes migrate along microtubules. Moreover, microinjection with antibodies that inhibit microtubule-associated motor proteins demonstrated that dynein and kinesin I participate in delivery of FR-containing endosomes to the perinuclear area and plasma membrane, respectively. Further, single particle tracking analysis revealed bidirectional motions of FR endosomes, mediated by dynein and kinesin motors associated with the same endosome. These experimental tools allowed us to use FR-containing endosomes to evaluate the impact of cholesterol on intracellular membrane trafficking. Lowering plasma membrane cholesterol by metabolic depletion or MbetaCD extraction was found to both increase FR-containing endosome motility and change endosome distribution from colocalization with Rab7 to colocalization with Rab4. These data provide evidence that cholesterol regulates intracellular membrane trafficking via modulation of the distribution of low molecular weight G-proteins that are adaptors for microtubule motors.

APC and GSK-3beta are involved in mPar3 targeting to the nascent axon and establishment of neuronal polarity.

SH Shi — 2006-07-21T17:01:59-00:00

Curr Biol, Vol. 14, No. 22. (23 November 2004), pp. 2025-2032.

In developing hippocampal neurons in culture, the evolutionarily conserved polarity complex mPar3/mPar6/aPKC selectively accumulates at the tip of one, and only one, of the immature neurites of a neuron and thus specifies the axon and generates neuronal polarity. How mPar3/mPar6 is enriched at the tip of the nascent axon, but not the dendrites, is not fully understood. Here, we report that mPar3 forms a complex with adenomatous polyposis coli (APC) and kinesin superfamily (KIF) 3A, proteins that move along microtubules. In polarizing hippocampal neurons, APC selectively accumulates at the nascent axon tip and colocalizes with mPar3. Expression of dominant-negative C terminus deletion mutants of APC or ectopic expression of APC leads to dislocalization of mPar3 and defects in axon specification and neuronal polarity. In addition to spatial polarization of APC, the selective inactivation of the GSK-3beta activity at the nascent axon tip is required for mPar3 targeting and polarization and establishing neuronal polarity. These results suggest that mPar3 is polarized in developing neurons through APC- and kinesin-mediated transport to the plus ends of rapidly growing microtubules at the nascent axon tip, a process that involves a spatially regulated GSK-3beta activity.

A novel sorting motif in the glutamate transporter excitatory amino acid transporter 3 directs its targeting in Madin-Darby canine kidney cells and hippocampal neurons.

C Cheng — 2006-07-20T15:16:08-00:00

J Neurosci, Vol. 22, No. 24. (15 December 2002), pp. 10643-10652.

The glutamate transporter excitatory amino acid transporter 3 (EAAT3) is polarized to the apical surface in epithelial cells and localized to the dendritic compartment in hippocampal neurons, where it is clustered adjacent to postsynaptic sites. In this study, we analyzed the sequences in EAAT3 that are responsible for its polarized localization in Madin-Darby canine kidney (MDCK) cells and neurons. Confocal microscopy and cell surface biotinylation assays demonstrated that deletion of the EAAT3 C terminus or replacement of the C terminus of EAAT3 with the analogous region in EAAT1 eliminated apical localization in MDCK cells. The C terminus of EAAT3 was sufficient to redirect the basolateral-preferring EAAT1 and the nonpolarized EAAT2 to the apical surface. Using alanine substitution mutants, we identified a short peptide motif in the cytoplasmic C-terminal region of EAAT3 that directs its apical localization in MDCK cells. Mutation of this sequence also impairs dendritic targeting of EAAT3 in hippocampal neurons but does not interfere with the clustering of EAAT3 on dendritic spines and filopodia. These data provide the first evidence that an identical cytoplasmic motif can direct apical targeting in epithelia and somatodendritic targeting in neurons. Moreover, our results demonstrate that the two fundamental features of the localization of EAAT3 in neurons, its restriction to the somatodendritic domain and its clustering near postsynaptic sites, are mediated by distinct molecular mechanisms.