Isch, C. | Search bioRxiv

bioRxiv2026

Dishevelled2 Condensates control Axon Initial Segment (AIS) assembly and function through MTCL1-Dependent microtubule recruitment.

In the epithelium, Planar Cell Polarity (PCP) signaling coordinates cellular organization along the plane of the tissue, perpendicular to the apicobasal axis. In neurons, polarity is instead expressed through the structural and molecular asymmetry that separates the axon from the somatodendritic compartment. Core PCP proteins are essential for neuronal development and function, and have been linked to neurodevelopmental disorders, including autism spectrum disorders and epilepsy. However, their precise roles in the establishment of neuronal polarity remain incompletely understood. Here, we identify Dishevelled2 (Dvl2), a central PCP signaling scaffold, as a novel component of the axon initial segment (AIS), the proximal axonal domain that maintains neuronal polarity and initiates action potentials. We show that Dvl2 defines, both in vivo and in cultured hippocampal neurons, a proximal subdomain positioned upstream of the major AIS marker Ankyrin-G (Ank-G) and is closely associated with microtubule bundles. Dvl2 is already detected during early axon specification, suggesting that it participates in the initial organization of the proximal axon. Functional depletion of Dvl proteins disrupts AIS architecture, reduces the accumulation of Ankyrin-G and other core AIS components, and impairs the organization of AIS microtubule bundles. These structural defects translate into altered neuronal excitability, with a reduced AIS-dependent component of the action potential. Mechanistically, we identify MTCL1 as a microtubule-associated partner that recruits Dvl2 from biomolecular condensate assemblies onto microtubules. This recruitment requires the DIX and PDZ domains of Dvl2. Notably, selective intracellular binders targeting the Dvl2 PDZ domain perturb Dvl2 condensate organization and compromise AIS formation. Together, our data identify Dvl2 as an organizer of the proximal AIS and reveal a Dvl2-MTCL1-dependent mechanism coupling condensate scaffolding, microtubule organization, and neuronal excitability.

neuroscience↗

bioRxiv2021

Structural and functional studies of the first tripartite protein complex at the Trypanosoma brucei flagellar pocket collar

The flagellar pocket (FP) is the only endo- and exocytic organelle in most trypanosomes and, as such, is essential throughout the life cycle of the parasite. The neck of the FP is maintained enclosed around the flagellum via the flagellar pocket collar (FPC). The FPC is a macromolecular cytoskeletal structure and is essential for the formation of the FP and cytokinesis. FPC biogenesis and structure are poorly understood, mainly due to the lack of information on FPC composition. To date, only two FPC proteins, BILBO1 and FPC4, have been characterized. BILBO1 forms a molecular skeleton upon which other FPC proteins can, theoretically, dock onto. We previously identified FPC4 as the first BILBO1 interacting partner and demonstrated that its C-terminal domain interacts with the BILBO1 N-terminal domain (NTD). Here, we report the characterization of a new FPC component and BILBO1 partner protein, BILBO2 (Tb927.6.3240) by yeast two-hybrid screen, bioinformatics, functional and structural studies. We show that BILBO2 colocalizes with BILBO1 and can modulate the shape of the BILBO1 filament by interacting with the BILBO1 EF-hand domains. Further, we demonstrate that BILBO1 and BILBO2 share a homologous NTD domain and that both domains interact with FPC4. We have determined a 1.9 [A] resolution crystal structure of the BILBO2 NTD in complex with the FPC4 BILBO1-binding domain. Together with mutational analyses, our studies reveal key residues for the function of the BILBO2 NTD and its interaction with FPC4 and evidenced a tripartite interaction between BILBO1, BILBO2, and FPC4. Our work sheds light on the first atomic structure of an FPC protein complex and represents a significant step in deciphering the FPC function in Trypanosoma brucei and other pathogenic kinetoplastids. Author summaryTrypanosomes belong to a group of zoonotic, protist, parasites that are found in Africa, Asia, South America, and Europe and are responsible for severe human and animal diseases. They all have a common structure called the flagellar pocket (FP). In most trypanosomes, all macromolecular exchanges between the trypanosome and the environment occur via the FP. The FP is thus essential for cell viability and evading the host immune response. We have been studying the flagellar pocket collar (FPC), an enigmatic macromolecular structure at the neck of the FP, and demonstrated its essentiality for the biogenesis of the FP. We demonstrated that BILBO1 is an essential protein of the FPC that interacts with other proteins including a microtubule-binding protein FPC4. Here we identify another bona fide FPC protein, BILBO2, so named because of close similarity with BILBO1 in protein organization and functional domains. We demonstrate that BILBO1 and BILBO2 share a common N-terminal domain involved in the interaction with FPC4, and illustrate a tripartite interaction between BILBO1, BILBO2, and FPC4. Our study also provides the first atomic view of two FPC components. These data represent an additional step in deciphering the FPC structure and function in T. brucei.

cell biology↗