In eukaryotes, protein secretion plays essential roles in intercellular communications and extracellular niche-building. Protein secretion generally requires a signal sequence that targets cargos to the canonical secretory pathway consisting of the endoplasmic reticulum (ER), the Golgi apparatus, plasma membrane, and vesicles moving between these compartments. However, cytoplasmic proteins lacking signal sequences (e.g. IL1β, Acb1, FGF2) have been detected and many have defined functions in the extracellular space, suggesting unconventional protein secretion (UcPS) via alternative pathways. In recent years, scientists have uncovered many new UcPS paradigms, reporting a plethora of mechanisms that collectively form a new field. The inaugural Cold Spring Harbor Asia (CSHA) conference on “ Molecular Mechanism and Physiology of Unconventional Secretion” is the first meeting to bring these researchers together, providing a collegial platform for information sharing at this exciting frontier of cell biology research.

The secretion of insulin and glucagon by pancreatic β and α cells, respectively, is critical for glucose homeostasis. While the insulin granule dynamics is well-characterized, the intracellular behavior of glucagon secretory granules (GSG) remains poorly understood. Here, we analyze the mobility of GSGs in α cells and insulin secretory granules (ISG) in β cells using spatiotemporal correlation spectroscopy and single-particle tracking (SPT), with a focus on the role of the cytoskeleton. Under basal conditions, SPT classification reveals that GSGs predominantly exhibit diffusive motion (57.6 ± 10%), with smaller fractions categorized as immobile (35.8 ± 10.6%) or drifted (6.6 ± 3%), resembling ISGs dynamics. By disrupting microtubules, we confirmed their role as active tracks for directed granule transport in both cell types. However, actin depolymerization, which enhances ISGs directed motion, has little effect on GSGs and instead increases their immobile fraction. This differential behavior persists under physiological secretory stimuli - high glucose for β cells and low glucose for α cells - indicating that, while both systems employ the cytoskeleton for secretion, α and β cells exhibit distinct recruitment and utilization patterns. These findings suggest that α and β cells may modulate granule dynamics differently to meet their specific secretory demands.

Adenoviral pVII proteins are multifunctional, highly basic, histone-like proteins that can bind to and transport the viral genome into the host cell nucleus. Despite the identification of several nuclear localization signals (NLSs) in the pVII protein of human adenovirus 5 (HAdV5), the mechanistic details of nuclear transport are largely unknown. Here we provide a full characterization of the nuclear import of precursor (Pre-) pVII protein from an ancient siadenovirus, frog siadenovirus 1 (FrAdV1) using a combination of structural, functional and biochemical approaches. Two strong NLSs (termed NLSa and NLSd) interact with importin (IMP)b and IMPa respectively, and are the main drivers of nuclear import. A weaker NLS (termed NLSb) also contributes, together with an additional signal (NLSc) which we found to be important for nucleolar targeting. Expression of Pre-pVII wild-type and NLS defective derivatives in the presence of selective inhibitors of different nuclear import pathways revealed that, unlike its human counterpart, FrAdV1 Pre-pVII nuclear import is dependent on IMPa/b and IMPb1. Clearly, AdVs evolved to maximise the nuclear import pathways for the pVII proteins, whose subcellular localization is the result of a complex process. Therefore, our results pave the way for an evolutionary comparison of the interaction of different AdVs with the host cell nuclear transport machinery.

The parasite Plasmodium falciparum causes the most severe form of malaria and to invade and replicate in red blood cells (RBCs), it exports hundreds of proteins across the encasing parasitophorous vacuole membrane (PVM) into this host cell. The exported proteins help modify the RBC to support rapid parasite growth and avoidance of the human immune system. Most exported proteins possess a conserved Plasmodium Export Element (PEXEL) motif with the consensus RxLxE/D/Q amino acid sequence, which acts as a proteolytic cleavage recognition site within the parasite’s endoplasmic reticulum (ER). Cleavage occurs after the P 1 L residue and is thought to help release the protein from the ER so it can be putatively escorted by the HSP101 chaperone to the parasitophorous vacuole space surrounding the intraerythrocytic parasite. HSP101 and its cargo are then thought to assemble with the rest of a Plasmodium Translocon for Exported proteins (PTEX) complex, that then recognises the xE/D/Q capped N terminus of the exported protein and translocates it across the vacuole membrane into the RBC compartment. Here, we present evidence that supports a dual role for the PEXEL’s conserved P 2’ position E/Q/D residue, firstly, for plasmepsin V cleavage in the ER, and secondly, for efficient PTEX mediated export across the PVM into the RBC. We also present evidence that the downstream ‘spacer’ region separating the PEXEL motif from the folded functional region of the exported protein controls cargo interaction with PTEX as well. The spacer must be of a sufficient length and permissive amino acid composition to engage the HSP101 unfoldase component of PTEX to be efficiently translocated into the RBC compartment.