Hinterlang, L. D. | Search bioRxiv

bioRxiv2025

Actin-related protein M1 (ARPM1) required for acrosome biogenesis and sperm function in mice

Actin-related proteins (Arps) are a superfamily of proteins which share sequence similarities with conventional actin and are involved in different cellular processes. Actin-related protein M1 (ARPM1) also known as actin-related protein T3 (ACTRT3) is a testis-enriched Arp which can be found in the perinuclear theca (PT) of murine round and elongating spermatids. ARPM1 forming a complex with Profilin 3 (PFN3) is lost in Pfn3-deficient sperm. We generated a mouse model deficient for Arpm1 and demonstrate that Arpm1-/- male mice are subfertile, with morphological aberrations of the acrosome. During spermiogenesis, defects become apparent in Cap phase of acrosome biogenesis when abnormal acrosomal granules are observed. Arpm1-deficiency causes deregulation of GM130 and TGN46 suggestive of defects in cis- and trans-Golgi trafficking required for acrosome development. Co-immunoprecipitation revealed that ARPM1 interacts with the PT-specific proteins ACTRT1, ACTRT2, ACTL7A and the sperm surface protein ZPBP, additionally to its already shown interaction with PFN3. We propose that ARPM1 acts as a structural component of the PT contributing to the cytoskeletal network connecting acrosome and nucleus. In addition, ARPM1 mediates the localization of ZPBP to enable fertilization and it tethers PFN3 to properly regulate Golgi-related acrosome development.

developmental biology↗

bioRxiv2024

Sensing endogenous RNA in living human cells using a CRISPR-activated protease

Most techniques used to detect specific mRNAs in eukaryotic cells require to extract nucleic acids and thereby kill the cells. A programmable sensor for monitoring endogenous transcripts in living cells, in contrast, would enable to enrich living cells based on a specific transcription or splicing event, and studying these cells by live microscopy or sequencing methods requiring intact cells. We have engineered CRISPR-READ, a live cell RNA detector based on the CRISPR-associated Lon protease CalpL and a cA4-producing Type III CRISPR system. Upon RNA-programmable RNA sensing, CRISPR-READ produces an orthogonal second messenger, which leads to the cleavage of a dual FRET / localization reporter compatible with FACS sorting and live microscopy. Using this genetically encoded sensing circuit as a readout for a genome-wide CRISPR perturbation screen, we identified an extended Type-I interferon signaling cascade; RNA-Seq on sensor-sorted cells enabled unbiased identification of correlated stochasticity in gene expression across single cells.

bioengineering↗