Transient directed motions of GABA(A) receptors in growth cones detected by a speed correlation index.

Single-molecule tracking of membrane proteins has become an important tool for investigating dynamic processes in live cells, such as cell signaling, membrane compartmentation or trafficking. The extraction of relevant parameters, such as interaction times between molecular partners or confinement-zone sizes, from the trajectories of single molecules requires appropriate statistical methods. Here we report a new tool, the speed correlation index, designed to detect transient periods of directed motion within trajectories of diffusing molecules. The ability to detect such events in a wide range of biologically relevant parameter values (speed, diffusion coefficient, and durations of the directed period) was first established on simulated data. The method was next applied to analyze the trajectories of quantum-dot-labeled GABA(A) receptors in nerve growth cones. The use of the speed correlation index revealed that the receptors had a "conveyor belt" type of motion due to temporary interactions ( approximately 4.0 s) between the receptors and the microtubules, leading to an average directed motion (velocity approximately 0.3 mum s(-1)) in the growth-cone membrane. Our observations point to the possibility of a cytoskeleton-dependent redistribution of the sensing molecules in the membrane, which could play a role in the modulation of the cell response to external signals.

[1]  A. Craig,et al.  Clustering of Gephyrin at GABAergic but Not Glutamatergic Synapses in Cultured Rat Hippocampal Neurons , 1996, The Journal of Neuroscience.

[2]  S. Gambhir,et al.  Quantum Dots for Live Cells, in Vivo Imaging, and Diagnostics , 2005, Science.

[3]  D. Bray,et al.  Signaling complexes: biophysical constraints on intracellular communication. , 1998, Annual review of biophysics and biomolecular structure.

[4]  Nicolas Destainville,et al.  Confined diffusion without fences of a g-protein-coupled receptor as revealed by single particle tracking. , 2003, Biophysical journal.

[5]  P. Haydon,et al.  Gephyrin Regulates the Cell Surface Dynamics of Synaptic GABAA Receptors , 2005, The Journal of Neuroscience.

[6]  K. Jacobson,et al.  Single-particle tracking: applications to membrane dynamics. , 1997, Annual review of biophysics and biomolecular structure.

[7]  M. Kneussel.  Postsynaptic scaffold proteins at non‐synaptic sites , 2005, EMBO reports.

[8]  N. Meilhac,et al.  Detection of confinement and jumps in single-molecule membrane trajectories. , 2005, Physical review. E, Statistical, nonlinear, and soft matter physics.

[9]  M. Poo,et al.  Nerve growth cone guidance mediated by G protein–coupled receptors , 2002, Nature Neuroscience.

[10]  K. Kalil,et al.  Touch and go: guidance cues signal to the growth cone cytoskeleton , 2005, Current Opinion in Neurobiology.

[11]  H. Qian,et al.  Single particle tracking. Analysis of diffusion and flow in two-dimensional systems. , 1991, Biophysical journal.

[12]  C. Waterman-Storer,et al.  Dual-wavelength fluorescent speckle microscopy reveals coupling of microtubule and actin movements in migrating cells , 2002, The Journal of cell biology.

[13]  K. Jacobson,et al.  Structural mosaicism on the submicron scale in the plasma membrane. , 1998, Biophysical journal.

[14]  M. Saxton,et al.  Single-particle tracking: models of directed transport. , 1994, Biophysical journal.

[15]  N. Brandon,et al.  GABAA-receptor-associated protein links GABAA receptors and the cytoskeleton , 1999, Nature.

[16]  C. Cohan,et al.  Focal loss of actin bundles causes microtubule redistribution and growth cone turning , 2002, The Journal of cell biology.

[17]  Daniel Choquet,et al.  Direct imaging of lateral movements of AMPA receptors inside synapses , 2003, The EMBO journal.

[18]  S. Dedhar,et al.  NGF-Induced Axon Growth Is Mediated by Localized Inactivation of GSK-3β and Functions of the Microtubule Plus End Binding Protein APC , 2004, Neuron.

[19]  Akihiro Kusumi,et al.  Relationship of lipid rafts to transient confinement zones detected by single particle tracking. , 2002, Biophysical journal.

[20]  A. Triller,et al.  Fast and reversible trapping of surface glycine receptors by gephyrin , 2001, Nature Neuroscience.

[21]  Thomas Schmidt,et al.  Single-molecule imaging of the H-ras membrane-anchor reveals domains in the cytoplasmic leaflet of the cell membrane. , 2007, Biophysical journal.

[22]  T. Yanagida,et al.  Single-Molecule Analysis of Chemotactic Signaling in Dictyostelium Cells , 2001, Science.

[23]  Laurent D. Cohen,et al.  Single quantum dot tracking based on perceptual Grouping using minimal paths in a spatiotemporal volume , 2005, IEEE Transactions on Image Processing.

[24]  K. Jacobson,et al.  Detection of temporary lateral confinement of membrane proteins using single-particle tracking analysis. , 1995, Biophysical journal.

[25]  Philippe Rostaing,et al.  Diffusion Dynamics of Glycine Receptors Revealed by Single-Quantum Dot Tracking , 2003, Science.

[26]  A. Triller,et al.  Strychnine-sensitive stabilization of postsynaptic glycine receptor clusters. , 1998, Journal of cell science.

[27]  M. Sheetz,et al.  Truncation mutants define and locate cytoplasmic barriers to lateral mobility of membrane glycoproteins. , 1994, Proceedings of the National Academy of Sciences of the United States of America.

[28]  S. McFarlane,et al.  GABA and development of the Xenopus optic projection. , 2002, Journal of neurobiology.

[29]  M. Igarashi,et al.  Signaling Pathway Downstream of GABAA Receptor in the Growth Cone , 1996, Journal of neurochemistry.

[30]  P. Forscher,et al.  Cytoskeletal remodeling during growth cone-target interactions , 1993, The Journal of cell biology.

[31]  R. Iino,et al.  Accumulation of anchored proteins forms membrane diffusion barriers during neuronal polarization , 2003, Nature Cell Biology.

[32]  A. Kusumi,et al.  Confined lateral diffusion of membrane receptors as studied by single particle tracking (nanovid microscopy). Effects of calcium-induced differentiation in cultured epithelial cells. , 1993, Biophysical journal.

[33]  R. Olsen,et al.  The Subcellular Distribution of GABARAP and Its Ability to Interact with NSF Suggest a Role for This Protein in the Intracellular Transport of GABAA Receptors , 2001, Molecular and Cellular Neuroscience.

[34]  Niels Galjart,et al.  Visualization of Microtubule Growth in Cultured Neurons via the Use of EB3-GFP (End-Binding Protein 3-Green Fluorescent Protein) , 2003, The Journal of Neuroscience.

[35]  R. Olsen,et al.  GABAA Receptor-Associated Protein Traffics GABAA Receptors to the Plasma Membrane in Neurons , 2004, The Journal of Neuroscience.