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1.
Curr Pharm Biotechnol ; 6(5): 405-14, 2005 Oct.
Article in English | MEDLINE | ID: mdl-16248814

ABSTRACT

Fluorescence correlation spectroscopy (FCS) and fluorescence cross-correlation spectroscopy (FCCS) are methods that extract information about a sample from the influence of thermodynamic equilibrium fluctuations on the fluorescence intensity. This method allows dynamic information to be obtained from steady state equilibrium measurements and its popularity has dramatically increased in the last 10 years due to the development of high sensitivity detectors and its combination with confocal microscopy. Using time-correlated single-photon counting (TCSPC) detection and pulsed excitation, information over the duration of the excited state can be extracted and incorporated in the analysis. In this short review, we discuss new methodologies that have recently emerged which incorporated fluorescence lifetime information or TCSPC data in the FCS and FCCS analysis. Time-gated FCS discriminates between which photons are to be incorporated in the analysis dependent upon their arrival time after excitation. This allows for accurate FCS measurements in the presence of fluorescent background, determination of sample homogeneity, and the ability to distinguish between static and dynamic heterogeneities. A similar method, time-resolved FCS can be used to resolve the individual correlation functions from multiple fluorophores through the different fluorescence lifetimes. Pulsed interleaved excitation (PIE) encodes the excitation source into the TCSPC data. PIE can be used to perform dual-channel FCCS with a single detector and allows elimination of spectral cross-talk with dual-channel detection. For samples that undergo fluorescence resonance energy transfer (FRET), quantitative FCCS measurements can be performed in spite of the FRET and the static FRET efficiency can be determined.


Subject(s)
Algorithms , Biopolymers/chemistry , Biopolymers/metabolism , Models, Biological , Photometry/methods , Photons , Spectrometry, Fluorescence/methods , Binding Sites , Biopolymers/analysis , Computer Simulation , Diffusion , Kinetics , Radiation Dosage , Radiometry/methods , Statistics as Topic , Time Factors
2.
Eur J Neurosci ; 10(5): 1574-80, 1998 May.
Article in English | MEDLINE | ID: mdl-9751130

ABSTRACT

Previous results of an in vitro guidance test, the stripe assay, have demonstrated the presence of a repulsive axon guidance activity for temporal retinal axons in the posterior part of the vertebrate optic tectum. Ephrin-A5 and Ephrin-A2 are ligands for the EphA subfamily of Eph receptor tyrosine kinases, which are expressed in overlapping gradients in the posterior part of the tectum. When recombinantly expressed, both proteins have been shown to guide retinal ganglion cell axons in the stripe assay. While these results suggest that Ephrin-A5 and Ephrin-A2 form part of the posterior repulsive guidance activity, they do not elucidate whether they are necessary components. Here we report that soluble forms of the ligands at nanomolar concentrations completely abolish this repulsive activity. Similar results were obtained with the soluble extracellular domain of EphA3, which is a receptor for Ephrin-A2 and Ephrin-A5, but not with the corresponding domain of EphB3, a receptor for the transmembrane class of Eph ligands. These experiments show that the repulsive axon guidance activity seen in the stripe assay is mediated by Ephrin-A ligands.


Subject(s)
Axons/metabolism , Receptor Protein-Tyrosine Kinases/metabolism , Retinal Ganglion Cells/metabolism , Signal Transduction/physiology , Cells, Cultured , Humans , Ligands , Proto-Oncogene Proteins c-myc/metabolism , Retinal Ganglion Cells/ultrastructure , Solubility
3.
EMBO J ; 16(6): 1258-67, 1997 Mar 17.
Article in English | MEDLINE | ID: mdl-9135142

ABSTRACT

Two ligands for Eph-related receptor tyrosine kinases, RAGS and ELF-1, have been implicated in the control of development of the retinotectal projection. Both molecules are expressed in overlapping gradients in the tectum, the target area of retinal ganglion cell axons. In two in vitro assays ELF-1 is shown to have a repellent axon guidance function for temporal, but apparently not for nasal axons. RAGS on the other hand is repellent for both types of axons, though to different degrees. Thus, RAGS and ELF-1 share some and differ in other properties. The biological activities of these molecules correlate with the strength of interaction with their receptors expressed on RGC axons. The meaning of these findings for guidance of retinal axons in the tectum is discussed.


Subject(s)
Axons/metabolism , DNA-Binding Proteins/metabolism , Retina/embryology , Retinal Ganglion Cells/metabolism , Transcription Factors/metabolism , Animals , Base Sequence , COS Cells , Chick Embryo , DNA Primers/genetics , DNA, Complementary/genetics , DNA-Binding Proteins/genetics , Ephrin-A2 , Gene Expression Regulation, Developmental , In Vitro Techniques , Mice , Nuclear Proteins , RNA/genetics , RNA/metabolism , Receptor Protein-Tyrosine Kinases/metabolism , Retina/cytology , Retina/metabolism , Superior Colliculi/cytology , Superior Colliculi/embryology , Superior Colliculi/metabolism , Transcription Factors/genetics , Transfection , Visual Pathways/cytology , Visual Pathways/embryology , Visual Pathways/metabolism
4.
Curr Opin Neurobiol ; 7(1): 75-80, 1997 Feb.
Article in English | MEDLINE | ID: mdl-9039788

ABSTRACT

The continued functional characterization of Eph-related receptors and ligands has provided further information toward an understanding of the mechanisms controlling the retinotectal projection. Recent in vivo analyses have strengthened the idea that Engrailed defines the positional identity of the tectum along the anteroposterior axis, possibly by regulating the expression of Eph family members.


Subject(s)
Axons/physiology , Membrane Proteins/physiology , Proteins/physiology , Retina/physiology , Visual Pathways/physiology , Animals , Ephrin-A2 , Ephrin-B2 , Superior Colliculi/physiology
5.
Development ; 123: 427-38, 1996 Dec.
Article in English | MEDLINE | ID: mdl-9007260

ABSTRACT

We have isolated mutants in the zebrafish Danio rerio that have defects in axonal connectivity between the retina and tectum. 5-day-old fish larvae were screened by labeling retinal ganglion cells with DiI and DiO and observing their axonal projections to and on the tectum. 82 mutations, representing 13 complementation groups and 6 single allele loci, were found that have defects in retinal ganglion cell axon pathfinding to the tectum. These pathfinding genes fall into five classes, based on the location of pathfinding errors between eye and tectum. In Class I mutant larvae (belladonna, detour, you-too, iguana, umleitung, blowout) axons grow directly to the ipsilateral tectal lobe after leaving the eye. Class II mutant larvae (chameleon, bashful) have ipsilaterally projecting axons and, in addition, pathfinding mistakes are seen within the eye. In Class III mutant larvae (esrom, tilsit, tofu) fewer axons than normal cross the midline, but some axons do reach the contralateral tectal lobe. Class IV mutant larvae (boxer, dackel, pinscher) have defects in axon sorting after the midline and retinal axons occasionally make further pathfinding errors upon reaching the contralateral tectal lobe. Finally, Class V mutant larvae (bashful, grumpy, sleepy, cyclops, astray) have anterior-posterior axon trajectory defects at or after the midline. The analysis of these mutants supports several conclusions about the mechanisms of retinal axon pathfinding from eye to tectum. A series of sequential cues seems to guide retinal axons to the contralateral tectal lobe. Pre-existing axon tracts seem not to be necessary to guide axons across the midline. The midline itself seems to play a central role in guiding retinal axons. Axons in nearby regions of the brain seem to use different cues to cross the ventral midline. Mutant effects are not all-or-none, as misrouted axons may reach their target, and if they do, they project normally on the tectum. The retinotectal pathfinding mutants reveal important choice points encountered by neuronal growth cones as they navigate between eye and tectum.


Subject(s)
Mutation , Retina/embryology , Superior Colliculi/embryology , Visual Pathways/embryology , Zebrafish/embryology , Zebrafish/genetics , Animals , Axons/physiology , Eye/embryology , Functional Laterality/genetics , Gene Expression Regulation, Developmental , Larva , Phenotype , Retina/anatomy & histology , Superior Colliculi/anatomy & histology , Visual Pathways/anatomy & histology , Zebrafish/anatomy & histology
6.
Curr Biol ; 6(11): 1497-502, 1996 Nov 01.
Article in English | MEDLINE | ID: mdl-8939610

ABSTRACT

BACKGROUND: The axons of retinal ganglion neurons from a precise topographic map in the optic tectum in the midbrain, and the guidance of retinal axons by directional cues in the tectum is crucial in this process. Several in vitro systems have been developed in order to identify the molecular basis of these directional cues. Temporal, but not nasal, retinal axons avoid posterior tectal membranes and grow on anterior membranes as a result of repellent guidance activities that are linked by glycosylphosphatidylinositol (GPI) anchors to the posterior membranes. A putative GPI-anchored repulsive guidance molecule with a molecular weight of 33 kDa has previously been characterized. Indirect results from experiments in vitro support the hypothesis that this 33 kDa molecule guides temporal retinal axons. RESULTS: To assess whether the 33 kDa protein is involved in axon guidance in vitro, we raised monoclonal antibodies against molecules that had been removed from tectal membranes by treatment with phosphatidylinositol-specific phospholipase C, which cleaves GPI anchors. A monoclonal immunoglobulin M, F3D4, recognized the 33 kDa molecule. In combination with chromophore-assisted laser inactivation, F3D4 caused a loss of the repellent activity from posterior tectal membranes in vitro. As a result, temporal retinal fibers were no longer repelled by posterior tectal membranes. This demonstrates that the F3D4 antigen, which we name RGM (repulsive guidance molecule) is involved in the guidance of retinal axons in an assay in vitro. In vivo, the expression of RGM increases from the anterior to the posterior pole of the optic tectum. CONCLUSIONS: These findings not only support the hypothesis that retinal axons are guided by gradients of repulsive guidance molecules but, in combination with earlier studies of receptor kinases and their ligands that act during guidance, argue for the presence of several repellent guidance molecules with similar functions in vitro and expression patterns in vivo.


Subject(s)
Axons/physiology , Coloring Agents/chemistry , Lasers , Proteins/metabolism , Retina/physiology , Rosaniline Dyes/chemistry , Saccharomyces cerevisiae Proteins , Animals , Antibodies, Monoclonal , Chick Embryo , Chitin Synthase , Ephrin-A2 , Fungal Proteins/metabolism , Mice , Mice, Inbred BALB C , Retina/radiation effects
7.
Curr Opin Genet Dev ; 6(4): 469-74, 1996 Aug.
Article in English | MEDLINE | ID: mdl-8791530

ABSTRACT

Recent studies have uncovered a bifunctional role of the diffusible axon guidance molecules netrin-1 and UNC-6 in that they attract some axons and steer others away simultaneously. Homology screens have extended the semaphorin and collapsin family to include at least 20 members, in both secreted and membrane-bound forms. Eph-related receptors and their membrane-bound ligands, the function of which have only been characterized poorly to date, have been added to the growing number of molecules involved in axon guidance and fasciculation.


Subject(s)
Axons/physiology , Caenorhabditis elegans Proteins , Glycoproteins/genetics , Helminth Proteins/genetics , Nerve Growth Factors/genetics , Nerve Tissue Proteins , Phosphoric Monoester Hydrolases/genetics , Receptor Protein-Tyrosine Kinases/genetics , Animals , Genes , Nervous System Physiological Phenomena , Netrin-1 , Netrins , Semaphorin-3A , Tumor Suppressor Proteins
8.
Eur J Cell Biol ; 69(4): 360-7, 1996 Apr.
Article in English | MEDLINE | ID: mdl-8741218

ABSTRACT

Indirect evidence supports a protective role of some EF-hand calcium-binding proteins against calcium-induced neurotoxicity. Little is known about how these proteins influence cytosolic calcium levels. After cloning the parvalbumin cDNA into an expression vector, teratocarcinoma cells (PCC7) were transfected. Parvalbumin-transfected and mock-transfected cells were loaded with the calcium indicator fura-2 and were exposed, in the same dish, to different concentrations of the calcium ionophore A23187 or to KCI. The results show that parvalbumin-transfected PCC7 cells had much better calcium buffering capacity than control cells.


Subject(s)
Calcium/metabolism , Parvalbumins/metabolism , Calcimycin/pharmacology , Fluorescent Dyes/pharmacology , Fura-2/pharmacology , Homeostasis , Ionophores/pharmacology , Parvalbumins/genetics , Potassium Chloride/pharmacology , Teratocarcinoma , Transfection , Tumor Cells, Cultured
9.
Curr Biol ; 5(11): 1255-6, 1995 Nov 01.
Article in English | MEDLINE | ID: mdl-8574582

ABSTRACT

Most monoclonal antibodies do not functionally inactivate the antigen they recognize. This limitation can be overcome by localized chromophore-assisted laser inactivation (CALI) of specific antibody-linked proteins.


Subject(s)
Antibodies, Monoclonal/chemistry , Antigens/chemistry , Lasers , Animals , Antibodies, Monoclonal/immunology , Antigens/immunology , Coloring Agents , Protein Denaturation
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