Biology subjects

Lucic, V.

Publications and source records attributed to Lucic, V..

7 recordsLinked to original sources

bioRxiv2025

Spatial organization of assemblies of protein complexes by colocalization analysis

The precise localization of multiple species of proteins and protein complexes organized in nanodomains and large, non-periodic protein assemblies has been shown to be important for cellular function. Cryo-electron tomography is uniquely suited to visualize such assemblies in situ, with single nanometer precision, together with their cellular environment. Here we present in detail and characterize a parameter-free, second order point pattern analysis method that is applicable to cellular cryo-electron tomograms, and provides quantitative and statistical characterization of the colocalization between two or more distinct groups of complexes, such as those that comprise protein assemblies. By numerical and analytical calculations, we show that this colocalization method can correctly detect and distinguish different forms of point pattern interactions between complexes, and therefore identify specific types of spatial distribution of complexes, which result from multiple biochemical interactions and diffusion in cellular environments. We also present image processing tasks that precede the colocalization to facilitate its applications to cellular junctions and other biological systems.

biophysics↗

bioRxiv2025

Cryo-correlative light and electron tomography of dopaminergic axonal varicosities reveals non-synaptic modulation of cortico-striatal synapses

Dopamine is an essential brain neuromodulator involved in reward and motor control. Dopaminergic (DA) neurons project to most brain areas, with particularly dense innervation in the striatum. DA varicosities bind to target striatal synapses and form dopamine hub synapses (DHS). However, the basic features of dopamine release sites are still largely unknown. Here we studied the ultrastructure of fluorescent DA and glutamatergic (GLU) synaptosomes isolated from the striatum of adult mice with cryo-correlative light and electron microscopy and cryo-electron tomography. We observed that DA synaptosomes display [~]10 times fewer vesicles than GLU ones. DA vesicles are bigger and less round. Vesicle organization at single nanometer scale indicates that most GLU synaptosomes have tethered and primed vesicles, indicative of a readily releasable pool, while only 39 % of DA synaptosomes have tethered vesicles, which appear not to be primed. In addition, GLU terminals contacted by DA terminals in DHS have more primed vesicles than others. While DA varicosities do not form genuine synapses, their adhesion to cortico-striatal synapses may convey a local regulation of synaptic release properties.

neuroscience↗

bioRxiv2024

Direct cryo-ET detection of native SNARE and Munc13 protein bridges using AI classification and preprocessing

Synaptic transmission requires Munc13 and SNARE proteins for synaptic vesicle priming and fusion. Cryo-electron tomography detected multiple types of Munc13- or SNARE-dependent dependent molecular bridges that tether synaptic vesicles to the presynaptic active zone plasma membrane. To integrate the molecular scenario with structural observations, we obtained de novo, in situ cryo-electron tomography averages of native, mammalian SNARE- and Munc13-dependent tethers. These provide direct evidence that Munc13 and a complex comprising SNARE proteins link synaptic vesicles to the active zone membrane. Furthermore, we determined the plausibility of different molecular compositions of tethers, placed constraints on their conformations and positioning, and proposed the existence of a complex downstream of Munc13 and upstream of SNARE complex formation. Because the detection and subtomogram averaging of membrane-bridging complexes is complicated by the presence of two lipid membranes and multiple protein species and conformations, we developed preprocessing methods and feature-based AI classifiers that outperformed standard methods.

neuroscience↗

bioRxiv2022

Synaptic vesicle-bound molecular bridges organize sequential vesicle states along parallel pathways

Synaptic vesicle tethering, priming, and neurotransmitter release require a coordinated action of multiple protein complexes. While physiological experiments, interaction data, and structural studies of purified systems were essential for our understanding of the function of the individual complexes involved, they cannot combine high structural detail with the unperturbed organization of complexes within cells to resolve how the actions of individual complexes integrate. We employed cryo-electron tomography to simultaneously image multiple presynaptic protein complexes and lipids at molecular resolution in their native composition, conformation and environment. Our results argue that tethers comprising proteins Munc13 and SNAP25 differentially and spatially confine vesicles with single nanometer precision, define vesicle tethering states, and provide molecular mechanisms that guide vesicles towards fusion, which includes molecular priming by conversion to SNARE complex-dependent tethers. These findings present an example of a cellular function performed by an extended molecular assembly comprising multiple, molecularly diverse complexes.

neuroscience↗

bioRxiv2022

Morphofunctional changes at the active zone during synaptic vesicle exocytosis

The fusion of synaptic vesicles (SVs) with the plasma membrane (PM) proceeds through intermediate steps that remain poorly resolved. Additionally, the effect of persistent high or low exocytosis activity on intermediate steps remains unknown. Through time-resolved cryo-electron tomography, we ordered events into a sequence. Following stimulation, proximal tethered SVs rapidly form additional tethers with the PM. Simultaneously, fusion initiation occurs by membrane curvature ( buckling) of the SV and PM. It is followed by the formation of a fusion pore, and the collapse of SV membrane. At this time, membrane-proximal, but not membrane-distal, vesicles lose their interconnections, allowing them to move towards the PM. Two mutations of SNAP-25 that arrests or disinhibit spontaneous release, respectively, both caused a loss of interconnectors, while the disinhibiting mutant also caused a loss of membrane proximal multiple-tethered SVs. Overall, tether formation and connector dissolution is triggered by stimulation and respond to the spontaneous fusion rate. These morphological observations likely correspond to the transition of SVs from one functional pool to another.

neuroscience↗

bioRxiv2020

Trans-synaptic assemblies link synaptic vesicles and neuroreceptors

Synaptic transmission is characterized by fast, tightly coupled processes and complex signaling path-ways that require a distinctly non-random spatial organization of their components. Nanoscale organization of synaptic proteins at glutamatergic synapses was suggested to regulate synaptic plasticity, the process underlying learning and memory. Specifically, direct colocalization of pre- and postsynaptic proteins implicated that the alignment of neurotransmitter release sites with neurotransmitter receptors enables maximal synaptic response. However, direct visualization and the mechanistic understanding of this alignment is lacking. Here we used cryo-electron tomography to visualize synaptic complexes in their native environment with the full complement of their interacting partners, synaptic vesicles and plasma membranes on 2-4 nanometer scale. The application of our recent template-free detection and classification procedure showed that tripartite trans-synaptic assemblies (subcolumns) link synaptic vesicles to postsynaptic receptors, and established that a particular displacement between directly interacting complexes characterizes subcolumns. Furthermore, we obtained de novo average structures of ionotropic glutamate receptors in their physiological composition, embedded in lipid membranes. The data presented support the hypothesis that synaptic function is carried by precisely organized trans-synaptic units. It complements superresolution findings and provides a framework for further exploration of synaptic and other large molecular assemblies that link different cells or cellular regions and may require weak or transient interactions to exert their function.

neuroscience↗

bioRxiv2018

Template-free detection and classification of heterogeneous membrane-bound complexes in cryo-electron tomograms

With faithful sample preservation and direct imaging of fully hydrated biological material, cryo-electron tomography (cryo-ET) provides an accurate representation of molecular architecture of cells. However, detection and precise localization of macromolecular complexes within cellular environments is aggravated by the presence of many molecular species and molecular crowding. We developed a template-free image processing procedure for accurate tracing of complex networks of densities in cryo-electron tomograms, a comprehensive and automated detection of heterogeneous membrane-bound complexes and an unsupervised classification. Applying this procedure to tomograms of intact cells and isolated endoplasmic reticulum (ER), we detected and classified small protein complexes like the ER protein translocons, which were not detected by other methods before. This classification provided sufficiently homogeneous particle sets and initial references to allow subsequent de novo subtomogram averaging. Therefore the procedure presented allows a comprehensive detection and a structural analysis of complexes in their native state. In addition, we present structural evidence that different ribosome-free translocon species are present at the ER membrane, determine their 3D structure, and show that they have different localization patterns forming nanodomains.

biophysics↗