Highlights d Calcium channels are organized in transient nanodomains d Ca V 2.1 channel mobility influences synaptic vesicle release d C-terminal splicing of Ca V 2.1 alters association with synaptic vesicles d Transient channel-scaffold interactions are relevant for shortterm plasticity

Fragile X syndrome (FXS) is the most common form of inherited mental retardation and is caused by the loss of function for Fragile X protein (FMRP), an RNA-binding protein thought to regulate synaptic plasticity by controlling the localization and translation of specific mRNAs. We have recently shown that FMRP is required to control the proliferation of the germline in Drosophila. To determine whether FMRP is also required for proliferation during brain development, we examined the distribution of cell cycle markers in dFmr1 brains compared with wild-type throughout larval development. Our results indicate that the loss of dFmr1 leads to a significant increase in the number of mitotic neuroblasts (NB) and BrdU incorporation in the brain, consistent with the notion that FMRP controls proliferation during neurogenesis. Developmental studies suggest that FMRP also inhibits neuroblast exit from quiescence in early larval brains, as indicated by misexpression of Cyclin E. Live imaging experiments indicate that by the third instar larval stage, the length of the cell cycle is unaffected, although more cells are found in S and G2/M in dFmr1 brains compared with wild-type. To determine the role of FMRP in neuroblast division and differentiation, we used Mosaic Analysis with a Repressible Marker (MARCM) approaches in the developing larval brain and found that single dFmr1 NB generate significantly more neurons than controls. Our results demonstrate that FMRP is required during brain development to control the exit from quiescence and proliferative capacity of NB as well as neuron production, which may provide insights into the autistic component of FXS.

Dynamic measurements of molecular machines can provide invaluable insights into their mechanism, but these measurements have been challenging in living cells. Here, we developed live-cell tracking of single fluorophores with nanometer spatial and millisecond temporal resolution in two and three dimensions using the recently introduced super-resolution technique MINFLUX. Using this approach, we resolved the precise stepping motion of the motor protein kinesin-1 as it walked on microtubules in living cells. Nanoscopic tracking of motors walking on the microtubules of fixed cells also enabled us to resolve the architecture of the microtubule cytoskeleton with protofilament resolution.

The cellular membrane is very heterogenous and enriched with high-density regions forming microdomains, as revealed by single particle tracking experiments. However the organization of these regions remain unexplained. We determine here the biophysical properties of these regions, when described as a basin of attraction. We develop two methods to recover the dynamics and local potential wells (field of force and boundary). The first method is based on the local density of points distribution of trajectories, which differs inside and outside the wells. The second method focuses on recovering the drift field that is convergent inside wells and uses the transient field to determine the boundary. Finally, we apply these two methods to the distribution of trajectories recorded from voltage gated calcium channels and phospholipid anchored GFP in the cell membrane of hippocampal neurons and obtain the size and energy of high-density regions with a nanometer precision.

No abstract

VGCCs are multisubunit complexes that play a crucial role in neuronal signaling. Auxiliary a2d subunits of VGCCs modulate trafficking and biophysical properties of the pore-forming a1 subunit and trigger excitatory synaptogenesis. Alterations in the expression level of a2d subunits were implicated in several syndromes and diseases, including chronic neuropathic pain, autism, and epilepsy. However, the contribution of distinct a2d subunits to excitatory/inhibitory imbalance and aberrant network connectivity characteristic for these pathologic conditions remains unclear. Here, we show that a2d1 overexpression enhances spontaneous neuronal network activity in developing and mature cultures of hippocampal neurons. In contrast, overexpression, but not downregulation, of a2d3 enhances neuronal firing in immature cultures, whereas later in development it suppresses neuronal activity. We found that a2d1 overexpression increases excitatory synaptic density and selectively enhances presynaptic glutamate release, which is impaired on a2d1 knockdown. Overexpression of a2d3 increases the excitatory synaptic density as well but also facilitates spontaneous GABA release and triggers an increase in the density of inhibitory synapses, which is accompanied by enhanced axonal outgrowth in immature interneurons. Together, our findings demonstrate that a2d1 and a2d3 subunits play distinct but complementary roles in driving formation of structural and functional network connectivity during early development. An alteration in a2d surface expression during critical developmental windows can therefore play a causal role and have a profound impact on the excitatory-to-inhibitory balance and network connectivity.

High voltage gated calcium channels (VGCCs) are composed of at least three subunits, one pore forming [Formula: see text]-subunit, an intracellular [Formula: see text]-variant, and a mostly extracellular [Formula: see text]-variant. Interactions between these subunits determine the kinetic properties of VGCCs. It is unclear whether these interactions are stable over time or rather transient. Here, we used single-molecule tracking to investigate the surface diffusion of [Formula: see text]- and [Formula: see text]-subunits at the cell surface. We found that [Formula: see text]-subunits show higher surface mobility than [Formula: see text]-subunits, and that they are only transiently confined together, suggesting a weak association between [Formula: see text]- and [Formula: see text]-subunits. Moreover, we observed that different [Formula: see text]-subunits engage in different degrees of association with the [Formula: see text]-subunit, revealing the tighter interaction of [Formula: see text] with [Formula: see text]. These data indicate a distinct regulation of the [Formula: see text] interaction in VGCC subtypes. We modeled their membrane dynamics in a Monte Carlo simulation using experimentally determined diffusion constants. Our modeling predicts that the ratio of associated [Formula: see text]- and [Formula: see text]-subunits mainly depends on their expression density and confinement in the membrane. Based on the different motilities of particular [Formula: see text]-subunit combinations, we propose that their dynamic assembly and disassembly represent an important mechanism to regulate the signaling properties of VGCC.

Dynamic measurements of molecular machines can provide invaluable insights into their mechanism, but have been challenging in living cells. Here, we developed live-cell tracking of single fluorophores with nanometer spatial and millisecond temporal resolution in 2D and 3D using the recently introduced super-resolution technique MINFLUX. This allowed us to resolve the precise stepping motion of the motor protein kinesin-1 as it walks on microtubules in living cells. In addition, nanoscopic tracking of motors on microtubule of fixed cells enabled us to resolve their spatial organization with protofilament resolution. Our approach will enable future in vivo studies of motor protein kinetics in complex environments and super-resolution mapping of dense microtubule arrays, and pave the way towards monitoring functional conformational changes of protein machines at high spatiotemporal resolution in living systems.