Insights into the Hair Cell Model: Unravelling the Trafficking Protein SorCS2 Localization Through Correlations with Rab11, Rab8, and Rab5

Abstract

Introduction: Hair cells in the organ of Corti are divided into inner and outer hair cell subtypes. The accurate alignment of stereociliary bundles is essential for the precise and sensitive transformation of mechanical stimuli into electrochemical signals. This alignment plays a critical role in activating auditory neurons and facilitating auditory coding. In a mouse line with profound deafness (Forge et al, 2017), mutations in the SorCS2 gene had a disrupted intrinsic polarity pathway, leading to defects in the formation of cochlear and vestibular hair cell bundles. Highlighting the critical role of SorCS2 in stereociliary bundle polarity during development. To identify the structures where it is likely to be expressed within cells and to understand SorCS2's life cycle, this study focussed in on its colocalization with Rab11, Rab8, and Rab5, essential GTPases involved in endocytic trafficking. Material and Methods: Protein subcellular distribution and molecular relationships were investigated in a simple genetically manipulable cell culture model system, mouse organ of Corti precursor cell line OC-2. Plasmid DNA constructs containing a GFP- tagged form of SorCS2, and mCherry-Rab11, Rab8, and Rab5, essential GTPases involved in endocytic trafficking, were transfected into cells. Confocal fluorescent microscopy was used analyse their cellular architecture. Confocal microscopy provided high-resolution images of the subcellular localization and spatial connections of tagged proteins. Colocalization image analysis was used to quantify the degree of protein colocalization. Results: The experimental results consistently demonstrate a strong positive correlation between SorCS2 and Rab11 which most likely localize in trans-Golgi network. The proteins' strong positive Pearson's correlation coefficient of 0.8 supports their close linkage and putative functional interactions in the Organ of Corti's cells. Using Pearson's correlation coefficient, Rab8 and SorCS2 co-localized in OC2 cells with mean 0.6 which most probably localize in TGN. In addition, images show cell morphological changes shaped like filopodia and lamellipodia. This study also examined SorCS2 and Rab5 colocalization in OC2 cells using Pearson's correlation coefficient which revealed that the two proteins have a mean average of 0.53, this suggests SorCS2 and Rab5 appear to be positively correlated and most likely localize transiently in the early endosome. Discussion: Our findings showed a strong positive correlation between Rab11 and SorCS2. Rab11 may regulate SorCS2 dynamics and cellular activities because of their co-localization and substantial correlations. Endosomal recycling and the trans-Golgi network may be where SorCS2 localises. SorCS2 and Rab8 colocalization were favourably associated by Pearson's coefficient. Confocal microscopy images confirmed this and showed a change in the cell's shape in the form of filopodia and lamellipodia, which may reveal the role of SorCS2 in cell shape, especially in the interaction between the reorganisation of actin filaments and microtubules. Moreover, this suggests that Rab8 and SorCS2 share the trans-Golgi network localization. Rab8 makes the polarisation function important. SorCS2 is probably present in early endosomes vesicles since Rab5 and SorCS2 are positively correlated in all samples. A diminished correlation in colocalization, as evidenced by the reduced Pearson's coefficient for Rab5, may suggest that the association between SorCS2-GFP and Rab5 is less stable or of shorter duration when compared to the other Rabs (Rab11 and Rab8). Conclusion: Protein localization techniques give a simplified view of SorCS2's location in a cell. Its colocalization with the Rab GTPases suggests that SorCS2 is mostly found in the endosomal recycling compartment but also in the trans-Golgi network and early endosome. These data show that SorCS2 localises more in the trans-Golgi network than in early endosomes. Previous studies have investigated the molecular processes of Rab11-dependent endocytic protein trafficking in polarised cells which may linked to the strong colocalization corelation. The findings imply that SorCS2 may affect hair cell polarity via Rab11 dependent recycling endosome protein trafficking. In addition, when Rab11 is active, it interacts with Rabin8, activating Rab8. Rab11 is found near primary cilia's base. Furthermore, Rab8 is present in filopodia, lamellipodia, protrusions, ruffles, and primary cilia, where it activates actin structures; inhibiting Rab8 decreases actin structure production. However, this project is about SorCS2 localization in a cultured cell model, therefore SorCS2's location and function in during development of hair cells within the inner ear need further study.