Quantal Transport Of Primordial Active Zones Via Ptvs

The results of several studies strongly support the idea that active zone components are transported in packages. Immunofluorescence localization revealed a punctate distribution and colocalization of Bassoon and Piccolo in axons of immature primary neurons in culture, consistent with an early association of these proteins with vesicles41,45. Likewise RIM showed a punctate distribution, but only an about 50% colocalization with Bassoon and Piccolo, suggesting that RIM might be associated with distinct organelles in addition42. The functional characterization of PTVs has been promoted by the use GFP-tagged versions of Bassoon, which has revealed to be a reliable marker for PTVs in live imaging studies performed on young neurons38,42, and for synapses in mature neurons46. For example, using GFP-Bassoon as a tool, PTVs were shown to be highly mobile in immature neurons, consistent with the notion that they serve as transport entities38,42. Combining these live imaging studies with immunofluoresence localization studies of endogenous proteins in quantitative analyses38,42 has generated the following emerging picture: There are small, mobile as well as larger, stable puncta positive for CAZ markers. The smaller puncta are typically negative, the larger puncta are typically positive for markers of mature synapses. The larger, synaptic puncta are on average two-fold brighter in fluorescence as compared to the smaller, mobile puncta. This would be consistent with mature active zones being built from material delivered by transport vesicles, and that typically more than one PTV generates an active zone.

Figure 16.4. The Active Zone Transport Vesicle Hypothesis: PTVs are Thought to Bud from the Trans-Golgi-Complex. At least Bassoon and Piccolo are packaged onto the cytoplasmic surface of PTVs already at this site, most likely at a sub-compartment of the trans-Golgi-network, and this event seems obligatory for presynaptic targeting of these proteins47. At the same site, PTVs may receive the entire complement of proteins (Figure 16.2). PTVs are supposed to travel along the axon via microtubule-based transport and are directed to nascent active zones by unknown signaling mechanisms. Fusion of PTVs (usually 2-3; refs. 38,42) with the axonal plasma membrane results in formation of an active zone. The topology of membrane fusion predicts that the CAZ proteins traveling on PTVs become located immediately underneath the plasma membrane, their contents are released, and transmembrane proteins become incorporated into the plasma membrane. SV precursors are also generated at the Golgi complex (left) and move along the axon along microtubules using independent pathways. (adapted from Dresbach, Altrock, Gundelfinger, Neuroforum 03/2003).

Figure 16.4. The Active Zone Transport Vesicle Hypothesis: PTVs are Thought to Bud from the Trans-Golgi-Complex. At least Bassoon and Piccolo are packaged onto the cytoplasmic surface of PTVs already at this site, most likely at a sub-compartment of the trans-Golgi-network, and this event seems obligatory for presynaptic targeting of these proteins47. At the same site, PTVs may receive the entire complement of proteins (Figure 16.2). PTVs are supposed to travel along the axon via microtubule-based transport and are directed to nascent active zones by unknown signaling mechanisms. Fusion of PTVs (usually 2-3; refs. 38,42) with the axonal plasma membrane results in formation of an active zone. The topology of membrane fusion predicts that the CAZ proteins traveling on PTVs become located immediately underneath the plasma membrane, their contents are released, and transmembrane proteins become incorporated into the plasma membrane. SV precursors are also generated at the Golgi complex (left) and move along the axon along microtubules using independent pathways. (adapted from Dresbach, Altrock, Gundelfinger, Neuroforum 03/2003).

But does an important postulate of the PTV hypothesis hold true, i.e., that a PTV delivers an entire set of active zone molecules? The answer appears to be yes based on further quantitative studies and modeling approaches: While there is a 1:2 ratio between CAZ-protein content of PTVs and synapses on average, both PTVs and mature synapses have a broad spectrum of individual fluorescence intensity levels. Taking into account the relative occurrence of individual fluorescence intensities of Bassoon associated with PTVs and synapses, reveals a striking correlation: The synaptic content of Bassoon can be accounted for by incorporation of integer numbers of fluorescence units into synapses. Specifically, two, three, and more rarely four fluorescence units of Bassoon appear to account for the Bassoon content of mature synapses in hippocampal primary neurons. The same applied to Piccolo and RIM. Moreover, such integer multiples calculated for Bassoon correctly predicted the synaptic content of RIM in the same experiment. These quantitative data strongly support the hypothesis that active zones may be assembled from unitary insertion of a small number of PTVs into the plasma membrane.

Further time-lapse imaging studies have provided additional support for this notion based on analysis of individual synapse formation events, as opposed to analysis of synapse populations. In these experiments, the occurrence of GFP-tagged Bassoon was imaged during formation of novel synapses. Synapse formation was monitored live by repeated application of the styryl dye FM4-64, which in the paradigms applied is taken up into and released from SVs at active synapses only. Mobile puncta of GFP-tagged Bassoon, presumably PTVs, were shown to arrive at sites where a novel synapse appeared in the course of the time lapse. Strikingly, when mobile puncta became stable at a new synapse, the fluorescence of the tagged protein increased stepwise at this site. In particular, this stepwise increase occurred from incorporation of two, three, four, or five puncta. These studies corroborate the active zone precursor hypothesis based on analysis of individual synapse formation events. Notably, several postsynaptic proteins were shown to be incorporated into newly forming synapses in a more gradual manner suggesting that postsynaptic assembly may differ from active zone assembly38. However, as discussed in detail by P.E. Washbourne (Chapter 14) various mechanisms may contribute to postsynaptic assembly. It should be noted that, to date, Bassoon and Piccolo seem to be the only CAZ components that are exclusively transported via PTVs, while others might be transported to the presynapse on various pathways. Moreover, CAZ formation via PTVs seems to be a major if not exclusive process during the major period of synaptogenesis during brain development. An important question for the future is: How is synapse formation and turnover mediated in the differentiated nervous system?

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