Secretion and function of Cln5 during the early stages of Dictyostelium development

Mutations in CLN5 cause neuronal ceroid lipofuscinosis (NCL), a currently untreatable neurodegenerative disorder commonly known as Batten disease. Several genetic models have been generated to study the function of CLN5, but one limitation has been the lack of a homolog in lower eukaryotic model systems. Our previous work revealed a homolog of CLN5 in the social amoeba Dictyostelium discoideum. We used a Cln5-GFP fusion protein to show that the protein is secreted and functions as a glycoside hydrolase in Dictyostelium. Importantly, we also revealed this to be the molecular function of human CLN5. In this study, we generated an antibody against Cln5 to show that the endogenous protein is secreted during the early stages of Dictyostelium development. Like human CLN5, the Dictyostelium homolog is glycosylated and requires this post-translational modification for secretion. Cln5 secretion bypasses the Golgi complex, and instead, occurs via an unconventional pathway linked to autophagy. Interestingly, we observed co-localization of Cln5 and GFP-Cln3 as well as increased secretion of Cln5 and Cln5-GFP in cln3^? cells. Loss of Cln5 causes defects in adhesion and chemotaxis, which intriguingly, has also been reported for Dictyostelium cells lacking Cln3. Finally, autofluorescence was detected in cln5^? cells, which is consistent with observations in mammalian systems. Together, our data support a function for Cln5 during the early stages of multicellular development, provide further evidence for the molecular networking of NCL proteins, and provide insight into the mechanisms that may underlie CLN5 function in humans.     

Cln3 function is linked to osmoregulation in a Dictyostelium model of Batten disease

Mutations in CLN3 cause a juvenile form of neuronal ceroid lipofuscinosis (NCL), commonly known as Batten disease. Currently, there is no cure for NCL and the mechanisms underlying the disease are not well understood. In the social amoeba Dictyostelium discoideum, the CLN3 homolog, Cln3, localizes predominantly to the contractile vacuole (CV) system. This dynamic organelle functions in osmoregulation, and intriguingly, osmoregulatory defects have been observed in mammalian cell models of CLN3 disease. Therefore, we used Dictyostelium to further study the involvement of CLN3 in this conserved cellular process. First, we assessed the localization of GFP-Cln3 during mitosis and cytokinesis, where CV system function is essential. GFP-Cln3 localized to the CV system during mitosis and cln3^? cells displayed defects in cytokinesis. The recovery of cln3^? cells from hypotonic stress and their progression through multicellular development was delayed and these effects were exaggerated when cells were treated with ammonium chloride. In addition, Cln3-deficiency reduced the viability of cells during hypotonic stress and impaired the integrity of spores. During hypertonic stress, Cln3-deficiency reduced cell viability and inhibited development. We then performed RNA sequencing to gain insight into the molecular pathways underlying the sensitivity of cln3^? cells to osmotic stress. This analysis revealed that cln3-deficiency upregulated the expression of tpp1A, the Dictyostelium homolog of human TPP1/CLN2. We used this information to show a correlated increase in Tpp1 enzymatic activity in cln3^? cells. In total, our study provides new insight in the mechanisms underlying the role of CLN3 in osmoregulation and neurodegeneration.     

Calmodulin‐mediated events during the life cycle of the amoebozoan Dictyostelium discoideum

This review focusses on the functions of intracellular and extracellular calmodulin, its target proteins and their binding proteins during the asexual life cycle of Dictyostelium discoideum. Calmodulin is a primary regulatory protein of calcium signal transduction that functions throughout all stages. During growth, it mediates autophagy, the cell cycle, folic acid chemotaxis, phagocytosis, and other functions. During mitosis, specific calmodulin‐binding proteins translocate to alternative locations. Translocation of at least one cell adhesion protein is calmodulin dependent. When starved, cells undergo calmodulin‐dependent chemotaxis to cyclic AMP generating a multicellular pseudoplasmodium. Calmodulin‐dependent signalling within the slug sets up a defined pattern and polarity that sets the stage for the final events of morphogenesis and cell differentiation. Transected slugs undergo calmodulin‐dependent transdifferentiation to re‐establish the disrupted pattern and polarity. Calmodulin function is critical for stalk cell differentiation but also functions in spore formation, events that begin in the pseudoplasmodium. The asexual life cycle restarts with the calmodulin‐dependent germination of spores. Specific calmodulin‐binding proteins as well as some of their binding partners have been linked to each of these events. The functions of extracellular calmodulin during growth and development are also discussed. This overview brings to the forefront the central role of calmodulin, working through its numerous binding proteins, as a primary downstream regulator of the critical calcium signalling pathways that have been well established in this model eukaryote. This is the first time the function of calmodulin and its target proteins have been documented through the complete life cycle of any eukaryote.