The use of fluorescence microscopy is central to cell biology in general, and essential to many fields (e.g., membrane traffic) that rely upon it to identify cellular locations of molecules under study and the extent to which they co-localize with others. Rigorous localization or co-localization data require quantitative image analyses that can vary widely between fields and laboratories. While most published data use two-dimensional images, there is an increasing appreciation for the advantages of collecting three-dimensional data sets. These include the ability to evaluate the entire cell and avoidance of focal plane bias. This is particularly important when imaging and quantifying changes in organelles with irregular borders and which vary in appearance between cells in a population, e.g., the Golgi. We describe a method developed for quantifying changes in signal intensity of one protein within any three-dimensional structure, defined by the presence of a different marker. We use as examples of this method the quantification of adaptor recruitment to transmembrane protein cargos at the Golgi though it can be directly applied to any site in the cell. Together, these advantages facilitate rigorous statistical testing of differences between conditions, despite variations in organelle structure, and we believe that this method of quantification of fluorescence data can be productively applied to a wide array of experimental questions.
image analysis; microscopy; immunofluorescence; confocal microscopy; wide field; membrane traffic; quantification; isosurface
There are numerous experimental approaches to identify the interaction networks of soluble proteins, but strategies for the identification of membrane protein interactomes remain limited. We discuss in detail the logic of an experimental design that led us to identify the interactome of a membrane protein of complex membrane topology, the calcium activated chloride channel Anoctamin 1/Tmem16a (Ano1). We used covalent chemical stabilizers of protein-protein interactions combined with magnetic bead immuno-affinity chromatography, quantitative SILAC mass-spectrometry and in silico network construction. This strategy led us to define a putative Ano1 interactome from which we selected key components for functional testing. We propose a combination of procedures to narrow down candidate proteins interacting with a membrane protein of interest for further functional studies.
membrane protein; Ano1; interactome; SILAC; epithelia; salivary gland
Numerous studies have shown that supraphysiological activation of AMPK could inhibit tumor growth. On the other hand, accumulating data also suggest that AMPK activity is required for tumor growth and migration. These findings suggest that physiological activation of AMPK is critical for tumor growth/migration, possibly through maintenance of ATP levels. Our recent study provides the first evidence that the maintenance of cellular NADPH homeostasis is the predominant mechanism by which AMPK promotes tumor cell survival and solid tumor formation. We showed that AMPK activation is required to maintain intracellular NADPH levels through the activation of fatty acid oxidation (FAO) or the inhibition of fatty acid synthesis (FAS) during glucose deprivation or matrix detachment respectively. Through these processes AMPK activation inhibits the rise in reactive oxygen species (ROS) levels and promotes metabolic adaptation in response to metabolic stress. This finding also provides a new therapeutic opportunity through targeting metabolic adaptation of cancer cells, either alone or in combination with conventional anti-cancer drugs that cause metabolic stress.
AMPK; LKB1; CaMKK2; ACC; NADPH; ROS; fatty acids; metabolic stress; cancer
We discuss here the variety of approaches that have been taken to inhibit different forms of endocytosis. Typically, both non-specific and specific chemical inhibitors of endocytosis are tried in order to “classify” entry of a new plasma membrane protein into one of the various types of endocytosis. This classification can be confirmed through genetic approaches of protein depletion or overexpression of mutants of known endocytosis machinery components. Although some new compounds have been designed to be selective in biochemical assays, we caution investigators to be alert to the unintended consequences that sometimes arise when these compounds are applied to intact cells.
endocytosis; pinocytosis; phagocytosis; clathrin-independent endocytosis; clathrin-mediated endocytosis; inhibitor; chemical inhibitor
Regulatory GTPases are often portrayed as binary molecular switches that control a wide range of cellular processes, including, but not limited to, the generation of second messengers (e.g., cAMP and inositol phosphates), intracellular traffic, cytoskeleton organization and cell proliferation. GEF stimulators and GAP inhibitors regulate the nucleotide-bound state of these proteins. Because of the relevance of GTPases and their regulators to human diseases, they comprise a major therapeutic target. Currently, most of the information about GTPase regulators comes from structure analyses. Such structural information is limited to certain conditions and does not necessarily reflect specificity or physiological activity. To address questions about specificity and mechanisms of action, kinetic and cell-based analyses of GTPase regulators is necessary. Here, we compare these two approaches in the context of regulators of Arf and heterotrimeric G-proteins.
GTPases; exchange factor; ADP-ribosylation factor; GDP; GEF
Exchange factors are enzymes that catalyze the exchange of GTP for GDP on guanine nucleotide binding proteins. Progress in understanding the molecular basis of action and the cellular functions of these enzymes has largely come from structural determinations (e.g., crystal structures) and studying effects on cells when expression levels of the exchange factors are perturbed or mutated exchange factors are expressed. Proportionally little effort has been expended on studying the kinetics of exchange; however, reaction rates are central to understanding enzymes. Here, we discuss the importance of kinetic analysis of exchange factors for guanine nucleotide binding proteins, with a focus on ADP-ribosylation factor (Arf) and heterotrimeric G proteins, for providing unique insights into molecular mechanisms and regulation as well as how kinetic analyses are used to complement other approaches.
G-protein couple receptor; guanine nucleotide binding protein; ADP-ribosylation factor; exchange factor; kinetics
Small GTPases of the Ras superfamily are important regulators of many cellular functions, including signal transduction, cytoskeleton assembly, metabolic regulation, organelle biogenesis and intracellular transport. Most GTPases act as binary switches, being “on” in the active, GTP-bound state and “off” in the inactive, GDP-bound state, and cycle between the two states with the aid of accessory proteins, referred to as guanine nucleotide exchange factors (GEFs) and GTPase-activating proteins (GAPs). This review will focus on the ADP-ribosylation factors (Arfs), a family of G-proteins that are essential regulators of carrier vesicle formation during vesicular transport. As for most other GTPases, the Arfs themselves are vastly outnumbered by the proteins that regulate them, and a major focus in the field has been to define the functional relationships between individual GEFs and GAPs and their substrates at the cellular level. Over the years, a variety of methods have been developed to measure GTPase activation in vitro and in vivo. In vitro analysis will be discussed in the accompanying article by Randazzo and colleagues. Here we will focus on cell- and tissue-based assays and their advantages/disadvantages relative to cell-free systems.
GTPase; Arf; GTP; GDP; GEF; GAP; effector; pulldown; FRET
The protein cargo transported by specific types of vesicles largely defines the different secretory trafficking pathways operating within cells. However, mole per mole the most abundant cargo contained within transport vesicles is not protein, but lipid. Taking a “lipid-centric” point-of-view, we examine the importance of lipid signaling, membrane lipid organization and lipid metabolism for vesicle transport during exocytosis in budding yeast. In fact, the essential requirement for some exocytosis regulatory proteins can be bypassed by making simple manipulations of the lipids involved. During polarized exocytosis the sequential steps required to generate post-Golgi vesicles and target them to the plasma membrane (PM) involves the interplay of several types of lipids that are coordinately linked through PI4P metabolism and signaling. In turn, PI4P levels are regulated by PI4P kinases, the Sac1p PI4P phosphatase and the yeast Osh proteins, which are homologs of mammalian oxysterol-binding protein (OSBP). Together these regulators integrate the transitional steps required for vesicle maturation directly through changes in lipid composition and organization.
polarized exocytosis; vesicle transport; lipid metabolism; sterols; PI4P; phosphoinositides; SAC1; oxysterol-binding proteins; Osh proteins; small GTPases
Retrograde trafficking mediates the transport of endocytic membranes from endosomes to the trans-Golgi network (TGN). Dysregulation of these pathways can result in multiple ailments, including late-onset Alzheimer disease. One of the key retrograde transport regulators, the retromer complex, is tightly controlled by many factors, including the C-terminal Eps15 homology domain (EHD) proteins. This mini-review focuses on recent findings and discusses the regulation of the retromer complex by EHD proteins and the novel EHD1 interaction partner, Rabankyrin-5 (Rank-5).
EHD1; EHD3; EH-domain; retromer; endocytic trafficking; recycling; retrograde transport; Rabankyrin-5
Cullin-RING-ligases (CRLs) comprise the largest class of multisubunit E3 ubiquitin ligases, which regulate a broad range of cellular processes. Cullin3 (Cul3) recently emerged as an important regulator of intracellular trafficking, in particular secretion and endosome maturation. Here we summarize and discuss possible functions and substrates of Cul3 in the endocytic system.
endocytosis; ubiquitin; Cullin3; Cul3; EGFR; influenza A virus
Exosomes, small secreted microvesicles, are implicated in intercellular communication in diverse cell types, transporting protein, lipid and nucleic acid cargo that impact the physiology of recipient cells. Besides the signaling function of exosomes they also serve as a mechanism to dispose obsolete cellular material.1 Particularly exciting is the involvement of exosomal communication in the nervous system, as this has important implications for brain development and function. The properties of exosomes are also beginning to entice the biomedical community since they represent potentially novel avenues for the targeted delivery of customized exosome cargo, such as miRNAs, during disease. Our findings implicating exosomes in trans-synaptic communication emerged from the serendipitous observation that at the Drosophila larval neuromuscular junction (NMJ) the release of a signaling molecule, Wnt1/Wingless (Wg) and its binding partner Evenness Interrupted (Evi)/Wntless (Wls)/Sprint (Srt), were released by motorneurons in association with vesicles, which we postulated to be exosomes.2 In our most recent paper3 using in vivo analysis at the Drosophila NMJ as well as in cultured insect cells we formally demonstrate that Evi rides in exosomes that are released to the extracellular space and identify some of the players involved in their release. In addition, a proteomic analysis of exosomes highlights novel potential function of exosomes.
neuromuscular junction; Drosophila; Evi/Wntless/GPR177/mig-14; retromer; local translation; exosome release; Wnt; Wingless; Rab11; Syntaxin 1A; exosomal proteome; RNA-binding proteins
Eukaryotic, prokaryotic and viral pathogens are known to interfere with signaling pathways of their host to promote their own survival and proliferation. Here, we present selected examples of modulation of PAK activity in human cells by both intracellular and extracellular pathogens, focusing on one eukaryotic pathogen, the human malaria parasite Plasmodium falciparum, two Gram-negative bacteria (Helicobacter pylori and Pseudomonas aeruginosa), and two viruses belonging to distinct groups, the lentivirus HIV and the orthomyxovirus Influenza virus A.
p21-activated kinase; pathogen; Plasmodium; Helicobacter; Pseudomonas; HIV; influenza
Protein kinases are versatile signaling molecules that are involved in the regulation most physiological responses. The p21-activated kinases (PAKs) can be activated directly by the small GTPases Rac and Cdc42 and are among the best characterized downstream effectors of these Rho proteins. The structure, substrate specificity and functional role of PAKS are evolutionarily conserved from protozoa to mammals. Vertebrate PAKs are particularly important for cytoskeletal remodeling and focal adhesion assembly, thereby contributing to dynamic processes such as cell migration and synaptic plasticity. This issue of Cellular Logistics focuses on the PAK family of kinases, with ten reviews written by researchers currently working in the field. Here in this introductory overview we highlight some of the most interesting recent discoveries regarding PAK biochemistry and biology. The reviews in this issue cover a range of topics including the atomic structures of PAK1 and PAK4, their role in animals as assessed by knockout studies, and how PAKs are likely to contribute to cancer and neurodegenerative diseases. The promise remains that PAK inhibitors will emerge that validate current pre-clinical studies suggesting that blocking PAK activity will positively contribute to human health.
PAK1 kinase is a crucial regulator of a variety of cellular processes, such as motility, cell division, gene transcription and apoptosis. Its deregulation is involved in several pathologies, including cancer, viral infection and neurodegenerative diseases. Due to this strong implication in human health, the complex network of signaling pathways centered on PAK1 is a subject of intensive investigations. This review summarizes the present knowledge on the multiple PAK1 intracellular localizations and on its shuttling between different compartments. The dynamics of PAK1 localization and activation are finely tuned by the cell and it is this tight control that underlies the capacity of PAK1 to participate in the regulation of many fundamental cell functions. Recently, PAK1 biosensors have been developed to visualize PAK1 activation in live cells. These new imaging tools should be of great help to better understand PAK1 biology and to conceive strategies for efficient and specific PAK1 inhibitors.
PAK1; actin cytoskeleton; motility; Rac1; cell imaging; fluorescence resonance energy transfer (FRET); spatiotemporal dynamics; functional microscopy; biosensor
PAKs 4, 5 and 6 are members of the group B family of p21-activated kinases. Among this group, PAK4 has been most extensively studied. While it has essential roles in embryonic development, in adults high levels of PAK4 are frequently associated with cancer. PAK4 is overexpressed in a variety of cancers, and the Pak4 gene is amplified in some cancers. PAK4 overexpression is sufficient to cause oncogenic transformation in cells and in mouse models. The tight connection between PAK4 and cancer make it a promising diagnostic tool as well as a potential drug target. The group B PAKs also have important developmental functions. PAK4 is important for many early developmental processes, while PAK5 and PAK6 play roles in learning and memory in mice. This chapter provides an overview of the roles of the group B PAKs in cancer as well as development, and includes a discussion of PAK mediated signaling pathways and cellular functions.
PAK4; group B PAKs; signal transduction; development; cancer
Transformation of a normal cell to a cancer cell is caused by mutations in genes that regulate proliferation, apoptosis, and invasion. Small GTPases such as Ras, Rho, Rac and Cdc42 orchestrate many of the signals that are required for malignant transformation. The p21-activated kinases (PAKs) are effectors of Rac and Cdc42. PAKs are a family of serine/threonine protein kinases comprised of six isoforms (PAK1–6), and they play important roles in cytoskeletal dynamics, cell survival and proliferation. They act as key signal transducers in several cancer signaling pathways, including Ras, Raf, NFκB, Akt, Bad and p53. Although PAKs are not mutated in cancers, they are overexpressed, hyperactivated or amplified in several human tumors and their role in cell transformation make them attractive therapeutic targets. This review discusses the evidence that PAK is important for cell transformation and some key signaling pathways it regulates. This review primarily discusses Group I PAKs (PAK1, PAK2 and PAK3) as Group II PAKs (PAK4, PAK5 and PAK6) are discussed elsewhere in this issue (by Minden).
cancer; amplification; PAK; p21 activated kinase; Rac; CDC42; protein kinase
Developmental cognitive deficits including X-linked mental retardation (XLMR) can be caused by mutations in P21-activated kinase 3 (PAK3) that disrupt actin dynamics in dendritic spines. Neurodegenerative diseases such as Alzheimer disease (AD), where both PAK1 and PAK3 are dysregulated, may share final common pathways with XLMR. Independent of familial mutation, cognitive deficits emerging with aging, notably AD, begin after decades of normal function. This prolonged prodromal period involves the buildup of amyloid-β (Aβ) extracellular plaques and intraneuronal neurofibrillary tangles (NFT). Subsequently region dependent deficits in synapses, dendritic spines and cognition coincide with dysregulation in PAK1 and PAK. Specifically proximal to decline, cytoplasmic levels of actin-regulating Rho GTPase and PAK1 kinase are decreased in moderate to severe AD, while aberrant activation and translocation of PAK1 appears around the onset of cognitive deficits. Downstream to PAK1, LIM kinase inactivates cofilin, contributing to cofilin pathology, while the activation of Rho-dependent kinase ROCK increases Aβ production. Aβ activation of fyn disrupts neuronal PAK1 and ROCK-mediated signaling, resulting in synaptic deficits. Reductions in PAK1 by the anti-amyloid compound curcumin suppress synaptotoxicity. Similarly other neurological disorders, including Huntington disease (HD) show dysregulation of PAKs. PAK1 modulates mutant huntingtin toxicity by enhancing huntingtin aggregation, and inhibition of PAK activity protects HD as well as fragile X syndrome (FXS) symptoms. Since PAK plays critical roles in learning and memory and is disrupted in many cognitive disorders, targeting PAK signaling in AD, HD and XLMR may be a novel common therapeutic target for AD, HD and XLMR.
Alzheimer disease; curcumin; PAK; ROCK; signaling pathways; synapses
The p21-activated kinases (PAKs) are downstream effectors of the small G-proteins of the Rac and cdc42 family and have been implicated as essential for cell proliferation and survival. Recent studies have also demonstrated the promise of PAKs as therapeutic targets in various types of cancers. The PAKs are divided into two major groups (group I and II) based on sequence similarities. Although the different roles the PAK groups might play are not well understood, recent efforts have focused on the identification of kinase inhibitors that can discriminate between the two groups. In this review these efforts and newly identified inhibitors will be described and future directions discussed.
The p21-activated kinases (PAKs) are a family of Ser/Thr protein kinases that are represented by six genes in humans (PAK 1–6), and are found in all eukaryotes sequenced to date. Genetic and knockdown experiments in frogs, fish and mice indicate group I PAKs are widely expressed, required for multiple tissue development, and particularly important for immune and nervous system function in the adult. The group II PAKs (human PAKs 4–6) are more enigmatic, but their restriction to metazoans and presence at cell-cell junctions suggests these kinases emerged to regulate junctional signaling. Studies of protozoa and fungal PAKs show that they regulate cell shape and polarity through phosphorylation of multiple cytoskeletal proteins, including microtubule binding proteins, myosins and septins. This chapter discusses what we know about the regulation of PAKs and their physiological role in different model organisms, based primarily on gene knockout studies.
Cdc42; Rac1; cytoskeleton; development
The p21-activated kinase (PAK) family plays a versatile role in cell signaling by forming a hub of interactions. PAKs bind the GTPases like RAC and CDC42. Their proline-rich motifs bind SH3 adaptor proteins such as PIX and NCK. PAKs display nuclear localization signal sites and a potential Integrin binding site. No fully complete structure of the PAKs has been published; partial 3D structures of the PAK family kinases include portions of the auto-inhibited PAK1, GTPase bound to small peptides from PAKs, and the kinase domains from PAK1 and PAK4–6 (with small ligands in a few cases). This review focuses on exploring the intermolecular interaction regions in these 3D structures and we offer insights on the missing regions in crystal structure of the auto-inhibited PAK1. Understanding and modulation of PAK intermolecular interactions can pave the way for PAK blockers and biosensors.
PAK; 3D structure; ligand binding site; affinity reagent; small-molecule inhibitor
p21-activated kinases are a family of highly conserved protein serine/threonine kinases that are increasingly recognized as playing essential roles in a variety of key signaling processes. Genetic analyses in mice, using constitutive or regulated gene disruption, have provided important new insights into PAK function. In this paper, we review the genetic analysis of all six PAK genes in mice. These data address the singular and redundant functions of the various PAK genes and suggest therapeutic possibilities for small molecule PAK inhibitors or activators.
p21-activated kinases; mouse models; signal transduction
Our work and others’ over the past few years have led to the identification of new roles of PAK1 in cardiac physiology, such as the regulation of cardiac ion channel and actomyosin function. More recent studies have revealed that PAK1-deficient mice were vulnerable to cardiac hypertrophy and readily progress to failure under sustained pressure overload and susceptible to ischemia/reperfusion injury. Our further study indicated that the PAK1 activator FTY720 was able to prevent this pressure overload-induced hypertrophy in wild-type mice without compromising their cardiac functions. A cardiac protective effect against ischemia/reperfusion injury by FTY720 was also observed in both rat and mouse models by us and others. Thus, these studies suggest that PAK1 is more important in the heart than previously thought, in particular a therapeutic potential of PAK1 activators. In the future, in-depth investigations are required to further substantiate our hypotheses on mechanisms for PAK1 function in the heart and to explore a therapeutic potential of FTY720 and other PAK1 activators in heart disease conditions.
PAK1; cardiac contractility; hypertrophic growth; ischemic injury