Biomedical subjects

Leon Lagnado

Publications and source records attributed to Leon Lagnado.

14 recordsLinked to original sources

PubMed2006

Clathrin-mediated endocytosis is the dominant mechanism of vesicle retrieval at hippocampal synapses.

The maintenance of synaptic transmission requires that vesicles be recycled after releasing neurotransmitter. Several modes of retrieval have been proposed to operate at small synaptic terminals of central neurons, including a fast "kiss-and-run" mechanism that releases neurotransmitter through a fusion pore. Using an improved fluorescent reporter comprising pHluorin fused to synaptophysin, we find that only a slow mode of endocytosis (tau = 15 s) operates at hippocampal synapses when vesicle fusion is triggered by a single nerve impulse or short burst. This retrieval mechanism is blocked by overexpression of the C-terminal fragment of AP180 or by knockdown of clathrin using RNAi, and it is associated with the movement of clathrin and vesicle proteins out of the synapse. These results indicate that clathrin-mediated endocytosis is the major, if not exclusive, mechanism of vesicle retrieval after physiological stimuli.

Animals↗

PubMed2006

Trimerisation is important for the function of clathrin at the mitotic spindle.

Clathrin is a triskelion consisting of three heavy chains each with an associated light chain. During mitosis, clathrin contributes to kinetochore fibre stability. As the N-terminal domain at the foot of each leg can bind to the mitotic spindle, we proposed previously a ;bridge hypothesis' wherein clathrin acts as a brace between two or three microtubules within a kinetochore fibre to increase fibre stability. Here, we have tested this hypothesis by replacing endogenous clathrin heavy chain in human cells with a panel of clathrin constructs. Mutants designed to abolish trimerisation were unable to rescue the mitotic defects caused by depletion of endogenous clathrin. By contrast, stunted triskelia with contracted legs could partially rescue normal mitosis. These results indicate that the key structural features of clathrin that are necessary for its function in mitosis are a trimeric molecule with a spindle interaction domain at each end, supporting the bridge hypothesis for clathrin function in mitosis.

Cells, Cultured↗

PubMed2005

Expansion of calcium microdomains regulates fast exocytosis at a ribbon synapse.

We investigated the Ca2+ signal regulating fast exocytosis at the ribbon synapse of retinal bipolar cells by using total internal reflection fluorescence microscopy to image fluorescent Ca2+ indicators and interference reflection microscopy to monitor exocytosis. Depolarization generated Ca2+ "microdomains" that expanded over the time scale during which the rapidly releasable pool (RRP) of vesicles was released (<40 ms). Replacing mobile Ca2+ buffers in the terminal with 10 mM EGTA prevented expansion of microdomains and decreased the number of rapidly releasable vesicles by a factor of 2. Conversely, decreasing the concentration of EGTA in the terminal to 0.1 mM increased the apparent width of a Ca2+ microdomain from 580 nm to 930 nm and increased the size of the RRP size by a factor of 1.5. The [Ca2+] over the area that the microdomain expanded was estimated to be 2-7 microM. These results indicate that vesicles within the RRP are located hundreds of nanometers from Ca2+ channels, and that fusion of these vesicles can be triggered by low micromolar levels of Ca2+. Variable distances between docked vesicles and Ca2+ channels at the active zone, therefore, provide an explanation for the heterogeneous release probability of vesicles comprising the RRP.

Animals↗

PubMed2005

Clathrin-dependent and clathrin-independent retrieval of synaptic vesicles in retinal bipolar cells.

Synaptic vesicles can be retrieved rapidly or slowly, but the molecular basis of these kinetic differences has not been defined. We now show that substantially different sets of molecules mediate fast and slow endocytosis in the synaptic terminal of retinal bipolar cells. Capacitance measurements of membrane retrieval were made in terminals in which peptides and protein domains were introduced to disrupt known interactions of clathrin, the AP2 adaptor complex, and amphiphysin. All these manipulations caused a selective inhibition of the slow phase of membrane retrieval (time constant approximately 10 s), leaving the fast phase (approximately 1 s) intact. Slow endocytosis after strong stimulation was therefore dependent on the formation of clathrin-coated membrane. Fast endocytosis occurring after weaker stimuli retrieves vesicle membrane in a clathrin-independent manner. All compensatory endocytosis required GTP hydrolysis, but only a subset of released vesicles were primed for fast, clathrin-independent endocytosis.

Adaptor Protein Complex 2↗

PubMed2005

Clathrin is required for the function of the mitotic spindle.

Clathrin has an established function in the generation of vesicles that transfer membrane and proteins around the cell. The formation of clathrin-coated vesicles occurs continuously in non-dividing cells, but is shut down during mitosis, when clathrin concentrates at the spindle apparatus. Here, we show that clathrin stabilizes fibres of the mitotic spindle to aid congression of chromosomes. Clathrin bound to the spindle directly by the amino-terminal domain of clathrin heavy chain. Depletion of clathrin heavy chain using RNA interference prolonged mitosis; kinetochore fibres were destabilized, leading to defective congression of chromosomes to the metaphase plate and persistent activation of the spindle checkpoint. Normal mitosis was rescued by clathrin triskelia but not the N-terminal domain of clathrin heavy chain, indicating that stabilization of kinetochore fibres was dependent on the unique structure of clathrin. The importance of clathrin for normal mitosis may be relevant to understanding human cancers that involve gene fusions of clathrin heavy chain.

Animals↗

PubMed2005

Ribbon synapses: anchors away for a fishy tale.

Genetic screens in flies and worms have long been a powerful way of identifying proteins that regulate synaptic transmission. A recent study of ribbon synapses in the retina of zebrafish is an excellent example of how this approach can now be applied to a vertebrate species.

Animals↗

PubMed2004

High mobility of vesicles supports continuous exocytosis at a ribbon synapse.

BACKGROUND: Most synapses release neurotransmitter as transient pulses, but ribbon synapses of sensory neurons support continuous exocytosis in response to maintained stimulation. We have investigated how the movement and retrieval of vesicles might contribute to continuous exocytosis at the ribbon synapse of retinal bipolar cells. RESULTS: Using a combination of total internal reflection fluorescence microscopy and fluorescence recovery after photobleaching, we found that the great majority of vesicles within 50-120 nm of the plasma membrane move in a random fashion with an effective diffusion coefficient of approximately 1.5 x 10(-2) microm(2) s(-1). Using confocal microscopy, we found that vesicles are similarly mobile across the whole terminal and that this motion is not altered by calcium influx or the actin cytoskeleton. We calculated that the cytoplasmic reservoir of approximately 300,000 vesicles would generate about 900 vesicle collisions/s against ribbons and 28,000 collisions/s against the surface membrane. The efficient resupply of vesicles to ribbons was confirmed by electron microscopy. A 1 min depolarization, releasing 500-1000 vesicles/s, caused a 70% reduction in the number of vesicles docked at the active zone without reducing the number of vesicles attached to ribbons or remote areas of the plasma membrane. These sites were not repopulated by retrieved vesicles because 80-90% of the recycled membrane was taken up into cisternae that pinched off from the surface. CONCLUSIONS: These results indicate that the random motion of cytoplasmic vesicles provides an efficient supply to the ribbon and plasma membrane and allows the maintenance of high rates of exocytosis without an equally rapid recycling of vesicles. The selective depletion of vesicles docked under ribbons suggests that the transfer of vesicles to the active zone limits the rate of exocytosis during maintained stimulation.

Animals↗

PubMed2003

Real-time measurement of exocytosis and endocytosis using interference of light.

We describe a new approach for making real-time measurements of exocytosis and endocytosis in neurons and neuroendocrine cells. The method utilizes interference reflection microscopy (IRM) to image surface membrane in close contact with a glass coverslip (the "footprint"). At the synaptic terminal of retinal bipolar cells, the footprint expands during exocytosis and retracts during endocytosis, paralleling changes in total surface area measured by capacitance. In chromaffin cells, IRM detects the fusion of individual granules as the appearance of bright spots within the footprint with spatial and temporal resolution similar to total internal reflection fluorescence microscopy. Advantages of IRM over capacitance are that it can monitor changes in surface area while cells are electrically active and it can be applied to mammalian neurons with relatively small synaptic terminals. IRM reveals that vesicles at the synapse of bipolar cells rapidly collapse into the surface membrane while secretory granules in chromaffin cells do not.

Animals↗

PubMed2003

Endocytosis at the synaptic terminal.

Exocytosis of neurotransmitter from a synaptic vesicle is followed by efficient retrieval of its constituent membrane and proteins. Real-time measurements indicate that fast and slow modes of retrieval operate in parallel at a number of presynaptic terminals. Two mechanisms can be distinguished by electron microscopy: clathrin-mediated retrieval of small vesicles and bulk retrieval of large cisternae. Methods that investigate the behaviour of individual vesicles have recently demonstrated a third route of retrieval: the rapid reversal of a pore-like connection between the vesicle and surface ('kiss-and-run'). Key aims for the future are to identify the molecules underlying different mechanisms of endocytosis at the synapse and the signals that select between them.

Animals↗

PubMed2003

Ribbon synapses.

Explore the source record for details and available documents.

Animals↗

PubMed2003

Exocytosis at the ribbon synapse of retinal bipolar cells studied in patches of presynaptic membrane.

The distribution of exocytic sites and ion channels in the synaptic terminal of retinal bipolar cells was investigated by measuring capacitance and conductance changes in cell-attached patches of presynaptic membrane. Patch depolarization evoked capacitance and conductance increases that were inhibited by blocking Ca(2+) influx or loading the terminal with EGTA. The increase in capacitance declined as the depolarization approached the reversal potential for Ca(2+), indicating that it was a result of Ca(2+)-dependent exocytosis. The conductance increase was caused by K(Ca) channels that were also activated by Ca(2+) influx. Two observations indicated that sites of exocytosis and endocytosis colocalized with clusters of Ca(2+) channels and K(Ca) channels; the initial rate of exocytosis was correlated with the activation of K(Ca) channels, and exocytosis did not occur in the 41% of patches lacking this conductance. Electron microscopy demonstrated that there were approximately 16 vesicles docked to the plasma membrane at each active zone marked by a ribbon, but vesicles were also attached to the rest of the membrane at a density of 1.5/microm(2). The density of ribbons was 0.10 +/- 0.02/microm(2), predicting that approximately 43% of cell-attached patches would lack an active zone. The density of Ca(2+) channel clusters assayed by capacitance and conductance responses was therefore similar to the density of ribbons. These results are consistent with the idea that Ca(2+) channel clusters were colocalized with ribbons but do not exclude the possibility that calcium channels also occurred at other sites. The wide distribution of vesicles docked to the plasma membrane suggests that exocytosis might also be triggered by the spread of Ca(2+) from Ca(2+) channel clusters.

Animals↗

PubMed2003

Bulk membrane retrieval in the synaptic terminal of retinal bipolar cells.

The mechanism of bulk membrane uptake at the synapse remains poorly defined, although exocytosis of synaptic vesicles is followed by compensatory membrane retrieval into both small vesicles and large cisternas or vacuoles. We investigated bulk retrieval in the presynaptic terminal of retinal bipolar cells. Fluorescence imaging of the membrane dye FM1-43 indicated that Ca2+-triggered exocytosis was followed by endocytosis into small vesicles and larger vacuoles that could be selectively labeled using large fluorescent dextrans. Disruption of actin filaments with cytochalasin D or latrunculin B inhibited the formation and transport of vacuoles, but exocytosis and endocytosis continued at normal rates. Bulk retrieval was linked to remodeling of the actin network, and both processes were inhibited by 2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one, an inhibitor of phosphatidylinositol 3-kinase (PI 3-kinase). The regulation of F-actin dynamics by Ca2+ and PI 3-kinase therefore played an important role in compensatory endocytosis at this synapse, but this role was confined to bulk membrane uptake. Capacitance measurements demonstrated that fast endocytosis and refilling of the rapidly releasable pool of vesicles were not dependent on F-actin or PI 3-kinase activity. The basic properties of bulk membrane retrieval at this synapse were very similar to macropinocytosis described in non-neural cells. Bulk retrieval did not play an essential role in maintaining the vesicle cycle during maintained stimulation, but we suggest that it may play a role in the structural plasticity of this synaptic terminal.

Actin Cytoskeleton↗

PubMed2002

Signal amplification: let's turn down the lights.

G protein activation by membrane-bound receptors initiates a chain reaction that amplifies cellular responses to external signals. In retinal photoreceptors, amplification by the phototransduction cascade is regulated by quickly switching off the visual pigment that acts as the receptor for light.

3',5'-Cyclic-GMP Phosphodiesterases↗

PubMed2002

Endogenous calcium buffers regulate fast exocytosis in the synaptic terminal of retinal bipolar cells.

Calcium-triggered exocytosis at the synapse is suppressed by addition of calcium chelators, but the effects of endogenous Ca(2+) buffers have not been tested. We find that 80% of Ca(2+) binding sites in the synaptic terminal of retinal bipolar cells were associated with mobile molecules that suppressed activation of Ca(2+)-sensitive K(+) channels with an efficiency equivalent to approximately 1.2 mM BAPTA. Removing these buffers caused a 30-fold increase in the number of vesicles released by Ca(2+) tail currents lasting approximately 0.5 ms and a 2-fold increase in the rapidly releasable pool of vesicles (RRP). The effects of BAPTA and EGTA indicate that vesicles comprising the RRP were docked at variable distances from Ca(2+) channels. We propose that endogenous Ca(2+) buffers regulate the size of the RRP by suppressing the release of vesicles toward the periphery of the active zone.

Animals↗