Fibrocystin/Polyductin releases a C-terminal fragment that translocates into mitochondria and suppresses cystogenesis.

Fibrocystin/Polyductin (FPC), encoded by PKHD1, is associated with autosomal recessive polycystic kidney disease (ARPKD), yet its precise role in cystogenesis remains unclear. Here we show that FPC undergoes complex proteolytic processing in developing kidneys, generating three soluble C-terminal fragments (ICDs). Notably, ICD15, contains a novel mitochondrial targeting sequence at its N-terminus, facilitating its translocation into mitochondria. This enhances mitochondrial respiration in renal epithelial cells, partially restoring impaired mitochondrial function caused by FPC loss. FPC inactivation leads to abnormal ultrastructural morphology of mitochondria in kidney tubules without cyst formation. Moreover, FPC inactivation significantly exacerbates renal cystogenesis and triggers severe pancreatic cystogenesis in a Pkd1 mouse mutant Pkd1V/V in which cleavage of Pkd1-encoded Polycystin-1 at the GPCR Proteolysis Site is blocked. Deleting ICD15 enhances renal cystogenesis without inducing pancreatic cysts in Pkd1V/V mice. These findings reveal a direct link between FPC and a mitochondrial pathway through ICD15 cleavage, crucial for cystogenesis mechanisms.

Mutually inhibitory Ras-PI(3,4)P2 feedback loops mediate cell migration.

Signal transduction and cytoskeleton networks in a wide variety of cells display excitability, but the mechanisms are poorly understood. Here, we show that during random migration and in response to chemoattractants, cells maintain complementary spatial and temporal distributions of Ras activity and phosphatidylinositol (3,4)-bisphosphate [PI(3,4)P2]. In addition, depletion of PI(3,4)P2 by disruption of the 5-phosphatase, Dd5P4, or by recruitment of 4-phosphatase INPP4B to the plasma membrane, leads to elevated Ras activity, cell spreading, and altered migratory behavior. Furthermore, RasGAP2 and RapGAP3 bind to PI(3,4)P2, and the phenotypes of cells lacking these genes mimic those with low PI(3,4)P2 levels, providing a molecular mechanism. These findings suggest that Ras activity drives PI(3,4)P2 down, causing the PI(3,4)P2-binding GAPs to dissociate from the membrane, further activating Ras, completing a positive-feedback loop essential for excitability. Consistently, a computational model incorporating such a feedback loop in an excitable network model accurately simulates the dynamic distributions of active Ras and PI(3,4)P2 as well as cell migratory behavior. The mutually inhibitory Ras-PI(3,4)P2 mechanisms we uncovered here provide a framework for Ras regulation that may play a key role in many physiological processes.

Function and regulation of the Arp2/3 complex during cell migration in diverse environments.

As the first de novo actin nucleator discovered, the Arp2/3 complex has been a central player in models of protrusive force production via the dynamic actin network. Here, we review recent studies on the functional role of the Arp2/3 complex in the migration of diverse cell types in different migratory environments. These findings have revealed an unexpected level of plasticity, both in how cells rely on the Arp2/3 complex for migration and other physiological functions and in the intricate modulation of the Arp2/3 complex by other actin regulators and upstream signaling cascades.

Novel protein Callipygian defines the back of migrating cells.

Asymmetric protein localization is essential for cell polarity and migration. We report a novel protein, Callipygian (CynA), which localizes to the lagging edge before other proteins and becomes more tightly restricted as cells polarize; additionally, it accumulates in the cleavage furrow during cytokinesis. CynA protein that is tightly localized, or "clustered," to the cell rear is immobile, but when polarity is disrupted, it disperses throughout the membrane and responds to uniform chemoattractant stimulation by transiently localizing to the cytosol. These behaviors require a pleckstrin homology-domain membrane tether and a WD40 clustering domain, which can also direct other membrane proteins to the back. Fragments of CynA lacking the pleckstrin homology domain, which are normally found in the cytosol, localize to the lagging edge membrane when coexpressed with full-length protein, showing that CynA clustering is mediated by oligomerization. Cells lacking CynA have aberrant lateral protrusions, altered leading-edge morphology, and decreased directional persistence, whereas those overexpressing the protein display exaggerated features of polarity. Consistently, actin polymerization is inhibited at sites of CynA accumulation, thereby restricting protrusions to the opposite edge. We suggest that the mutual antagonism between CynA and regions of responsiveness creates a positive feedback loop that restricts CynA to the rear and contributes to the establishment of the cell axis.

Assessment of development and chemotaxis in Dictyostelium discoideum mutants.

Studies using the social amoeba Dictyostelium discoideum have greatly contributed to the current understanding of the signaling network that underlies chemotaxis. Since directed migration is essential for normal D. discoideum multicellular development, mutants with chemotactic impairments are likely to have abnormal developmental morphologies. We have used multicellular development as a readout in a screen of mutants to identify new potential regulators of chemotaxis. In this chapter, we describe how mutants generated by restriction enzyme-mediated integration (REMI) are analyzed, from assessment of development to detailed characterization of 3',5'-cyclic adenosine monophosphate (cAMP)-induced responses. Two complementary approaches, plating cells either clonally on a bacterial lawn or as a population on non-nutrient agar, are used to evaluate multicellular development. Once mutants with aberrant developmental phenotypes are identified, their chemotaxis toward cAMP is assessed by both small population and micropipette assays. Furthermore, mutants are tested for defects in both general and specific signaling pathways by examining the recruitment of actin-binding LimE(Δcoil) or PIP3-binding PH domains to the plasma membrane in response to cAMP stimulation.

Eukaryotic chemotaxis: a network of signaling pathways controls motility, directional sensing, and polarity.

Chemotaxis, the directed migration of cells in chemical gradients, is a vital process in normal physiology and in the pathogenesis of many diseases. Chemotactic cells display motility, directional sensing, and polarity. Motility refers to the random extension of pseudopodia, which may be driven by spontaneous actin waves that propagate through the cytoskeleton. Directional sensing is mediated by a system that detects temporal and spatial stimuli and biases motility toward the gradient. Polarity gives cells morphologically and functionally distinct leading and lagging edges by relocating proteins or their activities selectively to the poles. By exploiting the genetic advantages of Dictyostelium, investigators are working out the complex network of interactions between the proteins that have been implicated in the chemotactic processes of motility, directional sensing, and polarity.