In vitro intercellular transfer of ONECUT homeoproteins

Details

Supervisors

Clotman, Frédéric

Faculty

Faculté des sciences

Degree label

Master [120] en biochimie et biologie moléculaire et cellulaire, à finalité approfondie

Abstract

The family of homeoprotein transcription factors, defined by a highly conserved 60-amino acid DNA-binding homeodomain, is known for their dual functionality, performing both intrinsic transcriptional regulation and non-cell-autonomous roles. Among them, the ONECUT (OC) proteins, distinguished by their additional cut domain and their atypical homeodomain, are key regulators in neuronal differentiation, spatial distribution, diversification, and identity maintenance, contributing to the proper development of the nervous system. Evidence from Oc mutant analyses has suggested that these proteins may travel between cells in vivo, a hypothesis further supported by in vitro studies demonstrating the intercellular transfer of human OC1 and OC2. This study aimed to investigate the intercellular transfer of mouse or human OC proteins, using Neuro-2a (N2a) cells as an in vitro model to mimic differentiating neurons. Using epifluorescence microscopy, we demonstrated that OC proteins can travel between cells and localize in the cytoplasm, in the perinuclear region and in the nucleus before and after transfer. Combined epifluorescence and confocal microscopy revealed that OC proteins form aggregates but do not localize in the Golgi apparatus, suggesting that their transfer is mediated by unconventional vesicular pathways. Additionally, no mitochondrial localization was observed in recipient cells, contrasting with studies on the Otx2 homeoprotein. Given the roles of OC proteins in neuronal identity specification, we investigated their potential to enhance the differentiation of immature N2a cells but found no detectable impact on generic differentiation. Furthermore, homeoprotein intercellular transfer is known to rely on two specific sequences: Sec, allowing secretion into the extracellular space, and Penetratin (Pen), which facilitates internalization into the recipient cell. OC proteins harbor a Sec-like sequence in their homeodomain and a Pen-like sequence in their cut domain. Using site-directed mutagenesis, we demonstrated that mutations in these sequences significantly impair intercellular transfer, highlighting their critical role in the process. Taken together, these findings provide new insights into the mechanisms underlying OC protein intercellular transfer and its potential implications for neuronal development coordination.