Abstract

Vimentin, a major intermediate filament protein, plays a crucial role in maintaining cellular structure and integrity. Beyond its structural function, Vimentin is increasingly recognized for its non-canonical roles, including the regulation of organelle positioning and dynamics. Mitochondria, essential for cellular energy and apoptosis, are known to interact with the Vimentin network, but the precise molecular determinants of this interaction remain poorly defined. This study aimed to identify the specific domain of the Vimentin protein responsible for direct binding to mitochondria. Using a purified system, we performed *in vitro* binding assays with isolated mitochondria and various recombinant fragments of Vimentin. Our results unequivocally demonstrate that the **N-terminal fragment of Vimentin** is both necessary and sufficient for the direct, high-affinity binding to the mitochondrial surface. This finding suggests that the highly conserved N-terminal head domain, which is typically involved in filament assembly and post-translational modifications, also serves as a critical tethering site for mitochondria. These results provide fundamental insight into the molecular mechanism governing Vimentin-mitochondria association, highlighting a novel regulatory point for mitochondrial distribution and function within the cell, which is particularly relevant in contexts such as cell migration and stress response.