Biology subjects

Basiashvili, T.

Publications and source records attributed to Basiashvili, T..

3 recordsLinked to original sources

bioRxiv2026

The molecular architecture of tunneling nanotubes

Tunneling nanotubes (TNTs) are thin intercellular bridges that mediate the exchange of proteins, organelles, and nucleic acids between neighboring cells. They are enriched in tumor cells, implicated in chemotherapy resistance, induced in models of aggregation-based diseases, and their formation is stimulated by viruses that use TNTs to enhance infection. Despite their broad relevance and therapeutic potential, TNT morphology and function remain poorly understood, owing to the absence of clear morphological criteria and the limitations of light microscopy. Here, we establish two complementary systems to study TNT formation and function: stimulation with the pseudorabies viral kinase US3 to model viral transmission, and treatment of acute monocytic leukemia THP-1 cells with daunorubicin to model chemotherapy resistance. Using live-cell imaging, we characterize cytoskeletal organization and bidirectional lysosome transport in both contexts, and apply cryo-correlative light and electron microscopy (cryo-CLEM) with cryogenic electron tomography (cryo-ET) to visualize TNTs in their native state at molecular resolution. We show that TNTs display a rich molecular architecture, comprising actin filaments, microtubules, intermediate filaments, active ribosomes, and diverse organelles including multivesicular bodies, autophagosomes, and lysosomes. Sub-nanometer microtubule reconstructions reveal mixed polarity within individual TNTs, suggesting that both connected cells actively contribute to TNT formation and cargo trafficking. This organization is conserved across both systems, implying that TNT biogenesis reflects a shared cellular program rather than a context-specific response. Our findings provide the first structural framework for TNTs, revealing an unexpectedly rich molecular architecture and laying the groundwork for understanding how TNTs orchestrate intercellular communication in disease.

cell biology↗

bioRxiv2025

In-cell cryo-electron tomography reveals differential effects of type I and type II kinase inhibitors on LRRK2 filament formation and microtubule association

Mutations in Leucine-Rich Repeat Kinase 2 (LRRK2) are a leading contributor to developing familial and idiopathic Parkinsons disease (PD). Most PD-causing LRRK2 mutations increase the kinase activity, leading to increased phosphorylation of Rab GTPases, disrupting vesicular trafficking, cytoskeletal dynamics, and autophagy. Under homeostatic conditions, the bulk of WT and PD-mutant LRRK2 is found in the cytosol. However, exogenously expressed LRRK2 can form microtubule-associated filaments that have been shown to affect molecular transport along microtubules in vitro. While the physiological relevance of microtubule binding has not been established yet, inhibitors being designed and tested as therapeutics have been shown to either promote or prevent filament formation of LRRK2. In this study, we examine the localization and resulting molecular organization of hyperactive LRRK2-I2020T, a common PD mutant, in cells treated with type I (MLi-2) or type II (GZD-824) kinase inhibitors. Treatment with a type I kinase inhibitor results in extensive LRRK2-I2020T decoration around microtubules and microtubule bundling. Stabilization of LRRK2-I2020T filaments by type I inhibitor treatment allowed us to build a full-length closed-kinase model of LRRK2-I2020T in its cellular environment. Conversely, treatment with a type II inhibitor resulted in minimal microtubule decoration by LRRK2-I2020T compared to Type I inhibitor treated cells. This study provides a structural framework for understanding how type I and type II kinase inhibitors differentially modulate LRRK2 filament formation, demonstrating that type I inhibitor treatment promotes a distinct filament architecture, whereas such assemblies are not observed with type II inhibitors.

biophysics↗

bioRxiv2024

Cryo-electron tomography reveals the microtubule-bound form of inactive LRRK2

Parkinsons Disease (PD) is the second most common neurodegenerative disorder. Mutations in leucine-rich repeat kinase 2 (LRRK2), a multi-domain protein containing both a kinase and a GTPase, are a leading cause of the familial form of PD. Pathogenic LRRK2 mutations increase LRRK2 kinase activity. While the bulk of LRRK2 is found in the cytosol, the protein associates with membranes where its Rab GTPase substrates are found, and under certain conditions, with microtubules. Integrative structural studies using single-particle cryo-electron microscopy (cryo-EM) and in situ cryo-electron tomography (cryo-ET) have revealed the architecture of microtubule-associated LRRK2 filaments, and that formation of these filaments requires LRRK2s kinase to be in the active-like conformation. However, whether LRRK2 can interact with and form filaments on microtubules in its autoinhibited state, where the kinase domain is in the inactive conformation and the N-terminal LRR domain covers the kinase active site, was not known. Using cryo-ET, we show that full-length LRRK2 can oligomerize on microtubules in its autoinhibited state. Both WT-LRRK2 and PD-linked LRRK2 mutants formed filaments on microtubules. While these filaments are stabilized by the same interfaces seen in the active-LRRK2 filaments, we observed a new interface involving the N-terminal repeats that were disordered in the active-LRRK2 filaments. The helical parameters of the autoinhibited-LRRK2 filaments are different from those reported for the active-LRRK2 filaments. Finally, the autoinhibited-LRRK2 filaments are shorter and less regular, suggesting they are less stable.

biophysics↗