Zita Balklava

Genome-wide analysis identifies a general requirement for polarity proteins in endocytic traffic

In a genome-wide RNA-mediated interference screen for genes required in membrane traffic — including endocytic uptake, recycling from endosomes to the plasma membrane, and secretion — we identified 168 candidate endocytosis regulators and 100 candidate secretion regulators. Many of these candidates are highly conserved among metazoans but have not been previously implicated in these processes. Among the positives from the screen, we identified PAR-3, PAR-6, PKC-3 and CDC-42, proteins that are well known for their importance in the generation of embryonic and epithelial-cell polarity. Further analysis showed that endocytic transport in Caenorhabditis elegans coelomocytes and human HeLa cells was also compromised after perturbation of CDC-42/Cdc42 or PAR-6/Par6 function, indicating a general requirement for these proteins in regulating endocytic traffic. Consistent with these results, we found that tagged CDC-42/Cdc42 is enriched on recycling endosomes in C. elegans and mammalian cells, suggesting a direct function in the regulation of transport.

A novel requirement for C. elegans Alix/ALX-1 in RME-1-mediated membrane transport

BACKGROUND Alix/Bro1p family proteins have recently been identified as important components of multivesicular endosomes (MVEs) and are involved in the sorting of endocytosed integral membrane proteins, interacting with components of the ESCRT complex, the unconventional phospholipid LBPA, and other known endocytosis regulators. During infection, Alix can be co-opted by enveloped retroviruses, including HIV, providing an important function during virus budding from the plasma membrane. In addition, Alix is associated with the actin cytoskeleton and might regulate cytoskeletal dynamics. RESULTS Here we demonstrate a novel physical interaction between the only apparent Alix/Bro1p family protein in C. elegans, ALX-1, and a key regulator of receptor recycling from endosomes to the plasma membrane, called RME-1. The analysis of alx-1 mutants indicates that ALX-1 is required for the endocytic recycling of specific basolateral cargo in the C. elegans intestine, a pathway previously defined by the analysis of rme-1 mutants. The expression of truncated human Alix in HeLa cells disrupts the recycling of major histocompatibility complex class I, a known Ehd1/RME-1-dependent transport step, suggesting the phylogenetic conservation of this function. We show that the interaction of ALX-1 with RME-1 in C. elegans, mediated by RME-1/YPSL and ALX-1/NPF motifs, is required for this recycling process. In the C. elegans intestine, ALX-1 localizes to both recycling endosomes and MVEs, but the ALX-1/RME-1 interaction appears to be dispensable for ALX-1 function in MVEs and/or late endosomes. CONCLUSIONS This work provides the first demonstration of a requirement for an Alix/Bro1p family member in the endocytic recycling pathway in association with the recycling regulator RME-1.

APP controls the formation of PI(3,5)P2 vesicles through its binding of the PIKfyve complex

Phosphoinositides are signalling lipids that are crucial for major signalling events as well as established regulators of membrane trafficking. Control of endosomal sorting and endosomal homeostasis requires phosphatidylinositol-3-phosphate (PI(3)P) and phosphatidylinositol-3,5-bisphosphate (PI(3,5)P2), the latter a lipid of low abundance but significant physiological relevance. PI(3,5)P2 is formed by phosphorylation of PI(3)P by the PIKfyve complex which is crucial for maintaining endosomal homeostasis. Interestingly, loss of PIKfyve function results in dramatic neurodegeneration. Despite the significance of PIKfyve, its regulation is still poorly understood. Here we show that the Amyloid Precursor Protein (APP), a central molecule in Alzheimer’s disease, associates with the PIKfyve complex (consisting of Vac14, PIKfyve and Fig4) and that the APP intracellular domain directly binds purified Vac14. We also show that the closely related APP paralogues, APLP1 and 2 associate with the PIKfyve complex. Whether APP family proteins can additionally form direct protein–protein interaction with PIKfyve or Fig4 remains to be explored. We show that APP binding to the PIKfyve complex drives formation of PI(3,5)P2 positive vesicles and that APP gene family members are required for supporting PIKfyve function. Interestingly, the PIKfyve complex is required for APP trafficking, suggesting a feedback loop in which APP, by binding to and stimulating PI(3,5)P2 vesicle formation may control its own trafficking. These data suggest that altered APP processing, as observed in Alzheimer’s disease, may disrupt PI(3,5)P2 metabolism, endosomal sorting and homeostasis with important implications for our understanding of the mechanism of neurodegeneration in Alzheimer’s disease.

The Amyloid Precursor Protein Controls PIKfyve Function

While the Amyloid Precursor Protein (APP) plays a central role in Alzheimer’s disease, its cellular function still remains largely unclear. It was our goal to establish APP function which will provide insights into APPs implication in Alzheimers disease. Using our recently developed proteo-liposome assay we established the interactome of APPs intracellular domain (known as AICD), thereby identifying novel APP interactors that provide mechanistic insights into APP function. By combining biochemical, cell biological and genetic approaches we validated the functional significance of one of these novel interactors. Here we show that APP binds the PIKfyve complex, an essential kinase for the synthesis of the endosomal phosphoinositide phosphatidylinositol-3,5-bisphosphate. This signalling lipid plays a crucial role in endosomal homeostasis and receptor sorting. Loss of PIKfyve function by mutation causes profound neurodegeneration in mammals. Using C. elegans genetics we demonstrate that APP functionally cooperates with PIKfyve in vivo. This regulation is required for maintaining endosomal and neuronal function. Our findings establish an unexpected role for APP in the regulation of endosomal phosphoinositide metabolism with dramatic consequences for endosomal biology and important implications for our understanding of Alzheimers disease.

The regulation of endocytosis by kinases: cell biology meets genomics

The mechanisms of signal transduction and vesicular transport have traditionally been studied in isolation, but recent studies make it clear that the two processes are inextricably linked. A new genome-wide analysis of human kinases using RNA interference shows an unexpected depth and complexity to the interactions between these processes.

A cell-permeable tool for analysing APP intracellular domain function and manipulation of PIKfyve activity.

In this work we developed and validated a cell permeable tool to study the intracellular function of a central molecule in Alzheimers disease, the amyloid precursor protein. We showed that it regulates the activity of the PIKfyve kinase complex.

Linking gene expression in the intestine to production of gametes through the phosphate transporter PITR-1 in Caenorhabditis elegans

Inorganic phosphate is an essential mineral for both prokaryotic and eukaryotic cell metabolism and structure. Its uptake into the cell is mediated by membrane-bound transporters and coupled to Na+ transport. Mammalian sodium-dependent Pi cotransporters have been grouped into three families NaPi-I, NaPi-II, and NaPi-III. Despite being discovered more than two decades ago, very little is known about requirements for NaPi-III transporters in vivo, in the context of intact animal models. Here we find that impaired function of the Caenorhabditis elegans NaPi-III transporter, pitr-1, results in decreased brood size and dramatically increased expression of vitellogenin by the worm intestine. Unexpectedly, we found that the effects of pitr-1 mutation on vitellogenin expression in the intestine could only be rescued by expression of pitr-1 in the germline, and not by expression of pitr-1 in the intestine itself. Our results indicate the existence of a signal from the germline that regulates gene expression in the intestine, perhaps linking nutrient export from the intestine to production of gametes by the germline.

Studying membrane trafficking in the worm C. elegans by RNA interference

A powerful approach to gain understanding of molecular machinery responsible for membrane trafficking is through inactivation of gene function by RNA interference (RNAi). RNAi-mediated gene silencing occurs when a double-stranded RNA is introduced into cells and targets a complementary mRNA for degradation. The subsequent lack of mRNA prevents the synthesis of the corresponding protein and ultimately causes depletion of a particular gene product from the cell. The effects of such depletion can then by analyzed by functional, morphological, and biochemical assays. RNAi-mediated knockdowns of numerous gene products in cultured cells of mammalian and other species origins have provided significant new insight into traffic regulation and represent standard approaches in current cell biology. However, RNAi in the multicellular nematode Caenorhabditis elegans model allows RNAi studies within the context of a whole organism, and thus provides an unprecedented opportunity to explore effects of specific trafficking regulators within the context of distinct developmental stages and diverse cell types. In addition, various transgenic C. elegans strains have been developed that express marker proteins tagged with fluorescent proteins to facilitate the analysis of trafficking within the secretory and endocytic pathways. This chapter provides a detailed description of a basic RNAi approach that can be used to analyze the function of any gene of interest in secretory and endosomal trafficking in C. elegans.