Sajeevan, A. | Search bioRxiv

bioRxiv2026

Extraction-dependent bone proteomics reveals distinct stable and dynamic protein modules during early post-exposure degradation

Bone is a highly durable biological tissue widely used in forensic, archaeological, and anthropological investigations; however, efficient protein recovery and understanding of protein stability over time remain major challenges in skeletal proteomics. Here, we systematically evaluated three bone protein extraction workflows and integrated them with data-independent acquisition (DIA) mass spectrometry to assess proteome coverage, reproducibility, and temporal protein dynamics under environmentally exposed conditions. Comparative analysis demonstrated that extraction strategy is a primary determinant of detectable proteome composition. EDTA-based demineralization followed by SDS extraction provided the deepest proteome coverage and highest reproducibility, whereas guanidine hydrochloride extraction preferentially enriched collagen and extracellular matrix proteins. In contrast, acid-based extraction yielded limited protein recovery. Temporal profiling of bone samples collected at 10 and 45 days post-exposure revealed two distinct protein classes. A temporally stable module, enriched in collagens and extracellular matrix proteins including COL1A2, COL5A2, BGN, SPARCL1, and NID2, exhibited minimal abundance change, indicating resistance to environmental degradation. In contrast, temporally dynamic proteins, enriched in mitochondrial, metabolic, and intracellular pathways such as ACO2, OGDH, PDHA1, ATP5PO, and PFKM, showed marked decline over time. These findings support a two-compartment model of bone protein preservation in which matrix-embedded proteins are preferentially retained while exposed intracellular proteins undergo progressive degradation. Collectively, this study establishes an integrated framework linking extraction methodology with temporal proteome stability and identifies candidate markers for skeletal preservation assessment and temporal biomarker development in forensic and archaeological applications.

systems biology↗

bioRxiv2025

High levels of Cdc42 GTPase underlie an all-or-none decision to fuse

Cdc42 is a Rho-family GTPase conserved across eukaryotes, where it plays essential roles in cell polarization. In single-celled yeast systems, Cdc42 is a key driver of symmetry breaking and polarized growth, forming zones of activity that locally recruit eRectors to organize the cytoskeleton and polarize secretion. Here we show that Cdc42 also functions in cell-cell fusion during Schizosaccharomyces pombe sexual reproduction but concentrates at the fusion site through mechanisms distinct from those proposed in Saccharomyces cerevisiae. Notably, the cdc42-mCherrySW allele (but not the cdc42-sfGFPSW allele), which is functional for cell polarization and has been used across organisms for dynamic studies, exhibits a strong fusion defect. These cells block fusion before cell wall digestion but after actin fusion focus formation, indicating that Cdc42 is required to translate the vesicle cluster into polarized cargo delivery. We trace the defect to instability of Cdc42-mCherrySW and demonstrate that cell fusion requires higher Cdc42 protein levels than mitotic polarized growth. Remarkably, by constructing an allelic series driving Cdc42 expression over a 5-fold range, we discover that polarized growth responds linearly to Cdc42 protein levels, whereas sexual reproduction exhibits a sharp switch-like response. Thus, the topology of the Cdc42 regulatory network is distinct for its polarization and mating functions.

cell biology↗