Leukocyte polarization in cell migration and immune interactions.

Cell migration plays a key role in a wide variety of biological phenomena. This process is particularly important for leukocyte function and the inflammatory response. Prior to migration leukocytes undergo polarization, with the formation of a lamellipodium at the leading edge and a uropod at the trailing edge. This cell shape allows them to convert cytoskeletal forces into net cell-body displacement. Leukocyte chemoattractants, including chemokines, provide directional cues for leukocyte motility, and concomitantly induce polarization. Chemoattractant receptors, integrins and other adhesion molecules, cytoskeletal proteins and intracellular regulatory molecules change their cellular localization during cell polarization. A complex system of signal transduction molecules, including tyrosine kinases, lipid kinases, second messengers and members of the Rho family of small GTPases is thought to regulate the cytoskeletal rearrangements underlying leukocyte polarization and migration. The elucidation of the mechanisms and signals that control this complex reorganization will lead to a better understanding of critical questions in cell biology of leukocyte migration and polarity.     

Crosstalk between small GTPases and polarity proteins in cell polarization

Cell polarization is crucial for the development of multicellular organisms, and aberrant cell polarization contributes to various diseases, including cancer. How cell polarity is established and how it is maintained remain fascinating questions. Conserved proteins of the partitioning defective (PAR), Scribble and Crumbs complexes guide the establishment of cell polarity in various organisms. Moreover, GTPases that regulate actin cytoskeletal dynamics have been implicated in cell polarization. Recent findings provide insights into polarization mechanisms and show intriguing crosstalk between small GTPases and members of polarity complexes in regulating cell polarization in different cellular contexts and cell types.     

Regulation of a late phase of T cell polarity and effector functions by Crtam

Spatial organization of cellular proteins plays an important role in establishment of cellular polarity to regulate cell division, differentiation, migration, and organogenesis. Activation of T cells by antigen-presenting cells (APCs) results in the formation of an immunological synapse (IS), assembly of a signaling scaffold at the T cell receptor (TCR) contact, cytoskeletal reorganization, and generation of second messengers within the first hours following intercellular contact. We demonstrate here that Crtam (class-I MHC-restricted T-cell associated molecule), an immunoglobulin-superfamily transmembrane protein, coordinates a signaling complex anchored by the Scrib polarity protein to establish a later phase of T cell polarity on a subset of CD4+ T cells >6 hours following activation. Maintenance of this late cellular polarity results in the ability of CD4+Crtam+ T cells to selectively produce more IFNgamma and IL22. Crtam engagement thus modulates signals many hours beyond the initial activation event and dynamically influences the adaptive immune response.     

Actin and Microtubules in Cell Motility: Which One is in Control?

The cytoskeleton is composed of three distinct elements: actin microfilaments, microtubules and intermediate filaments. The actin cytoskeleton is thought to provide protrusive and contractile forces, and microtubules to form a polarized network allowing organelle and protein movement throughout the cell. Intermediate filaments are generally considered the most rigid component, responsible for the maintenance of the overall cell shape. Cytoskeletal elements must be coordinately regulated for the cell to fulfill complex cellular functions, as diverse as cell migration, cell adhesion and cell division. Coordination between cytoskeletal elements is achieved by signaling pathways, involving common regulators such as the Rho guanosine-5'-triphosphatases (GTPases). Furthermore, evidence is now accumulating that cytoskeletal elements participate in regulating each other. As a consequence, although their functions seem well defined, they are in fact overlapping, with actin playing a role in membrane trafficking and microtubules being involved in the control of protrusive and contractile forces. This cytoskeletal crosstalk is both direct and mediated by signaling molecules. Cell motility is a well-studied example where the interplay between actin and microtubules appears bidirectional. This leads us to wonder which, if any, cytoskeletal element leads the way.     

Phosphatase Specificity and Pathway Insulation in Signaling Networks

Phosphatases play an important role in cellular signaling networks by regulating the phosphorylation state of proteins. Phosphatases are classically considered to be promiscuous, acting on tens to hundreds of different substrates. We recently demonstrated that a shared phosphatase can couple the responses of two proteins to incoming signals, even if those two substrates are from otherwise isolated areas of the network. This finding raises a potential paradox: if phosphatases are indeed highly promiscuous, how do cells insulate themselves against unwanted crosstalk? Here, we use mathematical models to explore three possible insulation mechanisms. One approach involves evolving phosphatase K_M values that are large enough to prevent saturation by the phosphatase’s substrates. Although this is an effective method for generating isolation, the phosphatase becomes a highly inefficient enzyme, which prevents the system from achieving switch-like responses and can result in slow response kinetics. We also explore the idea that substrate degradation can serve as an effective phosphatase. Assuming that degradation is unsaturatable, this mechanism could insulate substrates from crosstalk, but it would also preclude ultrasensitive responses and would require very high substrate turnover to achieve rapid dephosphorylation kinetics. Finally, we show that adaptor subunits, such as those found on phosphatases like PP2A, can provide effective insulation against phosphatase crosstalk, but only if their binding to substrates is uncoupled from their binding to the catalytic core. Analysis of the interaction network of PP2A’s adaptor domains reveals that although its adaptors may isolate subsets of targets from one another, there is still a strong potential for phosphatase crosstalk within those subsets. Understanding how phosphatase crosstalk and the insulation mechanisms described here impact the function and evolution of signaling networks represents a major challenge for experimental and computational systems biology.     

Tec kinases: shaping T-cell activation through actin

Following stimulation, T cells undergo marked actin-dependent changes in shape that are required for productive cellular interactions and movement during immune responses. Reorganization of the actin cytoskeletal is also necessary for the formation of an immunological synapse - the convergence of several signaling molecules at the plasma membrane that occurs after effective T-cell receptor (TCR) signaling. Much emerging evidence indicates that the Tec family of tyrosine kinases has a role in actin cytoskeleton reorganization. Specifically, T cells that lack or express mutant versions of the Tec kinase Itk show impaired TCR-induced actin polymerization, cell polarization and regulation of the signaling events involved in cytoskeletal reorganization. These data, as well as other findings, support roles for Tec kinases in actin cytoskeleton regulation.     

Protocadherin FAT1 binds Ena/VASP proteins and is necessary for actin dynamics and cell polarization

Cell migration requires integration of cellular processes resulting in cell polarization and actin dynamics. Previous work using tools of Drosophila genetics suggested that protocadherin fat serves in a pathway necessary for determining cell polarity in the plane of a tissue. Here we identify mammalian FAT1 as a proximal element of a signaling pathway that determines both cellular polarity in the plane of the monolayer and directed actin-dependent cell motility. FAT1 is localized to the leading edge of lamellipodia, filopodia, and microspike tips where FAT1 directly interacts with Ena/VASP proteins that regulate the actin polymerization complex. When targeted to mitochondrial outer leaflets, FAT1 cytoplasmic domain recruits components of the actin polymerization machinery sufficient to induce ectopic actin polymerization. In an epithelial cell wound model, FAT1 knockdown decreased recruitment of endogenous VASP to the leading edge and resulted in impairment of lamellipodial dynamics, failure of polarization, and an attenuation of cell migration. FAT1 may play an integrative role regulating cell migration by participating in Ena/VASP-dependent regulation of cytoskeletal dynamics at the leading edge and by transducing an Ena/VASP-independent polarity cue.     

The CBL–CIPK network mediates different signaling pathways in plants

The calcineurin B-like protein–CBL-interacting protein kinase (CBL–CIPK) signaling pathway in plants is a Ca²⁺-related pathway that responds strongly to both abiotic and biotic environmental stimuli. The CBL–CIPK system shows variety, specificity, and complexity in response to different stresses, and the CBL–CIPK signaling pathway is regulated by complex mechanisms in plant cells. As a plant-specific Ca²⁺ sensor relaying pathway, the CBL–CIPK pathway has some crosstalk with other signaling pathways. In addition, research has shown that there is crosstalk between the CBL–CIPK pathway and the low-K⁺ response pathway, the ABA signaling pathway, the nitrate sensing and signaling pathway, and others. In this paper, we summarize and review research discoveries on the CBL–CIPK network. We focus on the different modification and regulation mechanisms (phosphorylation and dephosphorylation, dual lipid modification) of the CBL–CIPK network, the expression patterns and functions of CBL–CIPK network genes, the responses of this network to abiotic stresses, and its crosstalk with other signaling pathways. We also discuss the technical research methods used to analyze the CBL–CIPK network and some of its newly discovered functions in plants.     

Centrosome positioning in polarized cells: Common themes and variations

The centrosome position is tightly regulated during the cell cycle and during differentiated cellular functions. Because centrosome organizes the microtubule network to coordinate both intracellular organization and cell signaling, centrosome positioning is crucial to determine either the axis of cell division, the direction of cell migration or the polarized immune response of lymphocytes. Since alteration of centrosome positioning seems to promote cell transformation and tumor spreading, the molecular mechanisms controlling centrosome movement in response to extracellular and intracellular cues are under intense investigation. Evolutionary conserved pathways involving polarity proteins and cytoskeletal rearrangements are emerging as common regulators of centrosome positioning in a wide variety of cellular contexts.     

The Rac-GAP Bcr is a novel regulator of the Par complex that controls cell polarity

Cell polarization is essential for many biological processes, including directed cell migration, and loss of polarity contributes to pathological conditions such as cancer. The Par complex (Par3, Par6, and PKCζ) controls cell polarity in part by recruiting the Rac-specific guanine nucleotide exchange factor T-lymphoma invasion and metastasis 1 (Tiam1) to specialized cellular sites, where Tiam1 promotes local Rac1 activation and cytoskeletal remodeling. However, the mechanisms that restrict Par-Tiam1 complex activity to the leading edge to maintain cell polarity during migration remain unclear. We identify the Rac-specific GTPase-activating protein (GAP) breakpoint cluster region protein (Bcr) as a novel regulator of the Par-Tiam1 complex. We show that Bcr interacts with members of the Par complex and inhibits both Rac1 and PKCζ signaling. Loss of Bcr results in faster, more random migration and striking polarity defects in astrocytes. These polarity defects are rescued by reducing PKCζ activity or by expressing full-length Bcr, but not an N-terminal deletion mutant or the homologous Rac-GAP, Abr, both of which fail to associate with the Par complex. These results demonstrate that Bcr is an integral member of the Par-Tiam1 complex that controls polarized cell migration by locally restricting both Rac1 and PKCζ function.     

Spatial phosphoprotein profiling reveals a compartmentalized extracellular signal-regulated kinase switch governing neurite growth and retraction

Brain development and spinal cord regeneration require neurite sprouting and growth cone navigation in response to extension and collapsing factors present in the extracellular environment. These external guidance cues control neurite growth cone extension and retraction processes through intracellular protein phosphorylation of numerous cytoskeletal, adhesion, and polarity complex signaling proteins. However, the complex kinase/substrate signaling networks that mediate neuritogenesis have not been investigated. Here, we compare the neurite phosphoproteome under growth and retraction conditions using neurite purification methodology combined with mass spectrometry. More than 4000 non-redundant phosphorylation sites from 1883 proteins have been annotated and mapped to signaling pathways that control kinase/phosphatase networks, cytoskeleton remodeling, and axon/dendrite specification. Comprehensive informatics and functional studies revealed a compartmentalized ERK activation/deactivation cytoskeletal switch that governs neurite growth and retraction, respectively. Our findings provide the first system-wide analysis of the phosphoprotein signaling networks that enable neurite growth and retraction and reveal an important molecular switch that governs neuritogenesis.     

Crosstalk of cell polarity signaling pathways

Cell polarity, the asymmetric organization of cellular components along one or multiple axes, is present in most cells. From budding yeast cell polarization induced by pheromone signaling, oocyte polarization at fertilization to polarized epithelia and neuronal cells in multicellular organisms, similar mechanisms are used to determine cell polarity. Crucial role in this process is played by signaling lipid molecules, small Rho family GTPases and Par proteins. All these signaling circuits finally govern the cytoskeleton, which is responsible for oriented cell migration, cell shape changes, and polarized membrane and organelle trafficking. Thus, typically in the process of cell polarization, most cellular constituents become polarized, including plasma membrane lipid composition, ion concentrations, membrane receptors, and proteins in general, mRNA, vesicle trafficking, or intracellular organelles. This review gives a brief overview how these systems talk to each other both during initial symmetry breaking and within the signaling feedback loop mechanisms used to preserve the polarized state.     

Making sense of hormone crosstalk during plant immune responses

In response to biotic stress, crosstalk between plant hormonal signaling pathways prioritizes defense over other cellular functions. Some plant pathogens take advantage of this regulatory system by mimicking hormones that interfere with host immune responses to promote virulence. Here we discuss the various roles that crosstalk may play in response to pathogens with different infection strategies.     

Cytoskeletal network in colon cancer: from genes to clinical application

Colorectal cancer arises from well-defined sequential steps characterised by distinct genetic events. Abnormalities in the expression and functional activity of cell adhesion molecules are implicated in the development and progression of the majority of colorectal cancers. Intercellular (e.g. E-cadherin/catenin complex) and cell-matrix (e.g. integrins) adhesion molecules are more than just cementing substances but regulate cell polarity, differentiation, proliferation, migration and invasion. Many of these cellular events are mediated through their intimate association with the actin cytoskeletal network. A dynamic actin cytoskeleton characterises normal epithelial cells and polymerisation and depolymerisation of actin filaments enables cell shape to change during migration and mitosis. In colorectal cancer, cells lose actin cytoskeletal organisation and normal cell adhesion when they become invasive. Future investigations should allow the unravelling of new cytoskeletal network functions in tumour biology and may lead to the development of novel therapeutic strategies based on the manipulation of its associated molecules.     

Central roles of small GTPases in the development of cell polarity in yeast and beyond.

Summary: The establishment of cell polarity is critical for the development of many organisms and for the function of many cell types. A large number of studies of diverse organisms from yeast to humans indicate that the conserved, small-molecular-weight GTPases function as key signaling proteins involved in cell polarization. The budding yeast Saccharomyces cerevisiae is a particularly attractive model because it displays pronounced cell polarity in response to intracellular and extracellular cues. Cells of S. cerevisiae undergo polarized growth during various phases of their life cycle, such as during vegetative growth, mating between haploid cells of opposite mating types, and filamentous growth upon deprivation of nutrition such as nitrogen. Substantial progress has been made in deciphering the molecular basis of cell polarity in budding yeast. In particular, it becomes increasingly clear how small GTPases regulate polarized cytoskeletal organization, cell wall assembly, and exocytosis at the molecular level and how these GTPases are regulated. In this review, we discuss the key signaling pathways that regulate cell polarization during the mitotic cell cycle and during mating.     

Planar polarity in the Drosophila eye: a multifaceted view of signaling specificity and cross-talk

Functional tissues not only polarize their epithelia in the apical-basolateral axis, but also often display a polarity within the plane of the epithelium. In Drosophila, all adult structures are derived from epithelia called imaginal discs and display planar polarization; the eye and the wing are particularly well suited for analysis. Studies of their polarization have identified several conserved genes that regulate both nuclear signaling and cytoskeletal architecture. In particular, the Frizzled (Fz) receptor has been identified as a key component of polarity establishment in all tissues. The Fz signaling pathway and associated events are beginning to be unraveled, shedding light on a novel Wnt/Fz signaling cascade.     

A genetic algorithm-based boolean delay model of intracellular signal transduction in inflammation

Background

Signal transduction is the major mechanism through which cells transmit external stimuli to evoke intracellular biochemical responses. Understanding relationship between external stimuli and corresponding cellular responses, as well as the subsequent effects on downstream genes, is a major challenge in systems biology. Thus, a systematic approach to integrate experimental data and qualitative knowledge to identify the physiological consequences of environmental stimuli is needed.

Results

In present study, we employed a genetic algorithm-based Boolean model to represent NF-κB signaling pathway. We were able to capture feedback and crosstalk characteristics to enhance our understanding on the acute and chronic inflammatory response. Key network components affecting the response dynamics were identified.

Conclusions

We designed an effective algorithm to elucidate the process of immune response using comprehensive knowledge about network structure and limited experimental data on dynamic responses. This approach can potentially be implemented for large-scale analysis on cellular processes and organism behaviors.

     

Neuronal polarity and trafficking

Among the most morphologically complex cells, neurons are masters of membrane specialization. Nowhere is this more striking than in the division of cellular labor between the axon and the dendrites. In morphology, signaling properties, cytoskeletal organization, and physiological function, axons and dendrites (or more properly, the somatodendritic compartment) are radically different. Such polarization of neurons into domains specialized for either receiving (dendrites) or transmitting (axons) cellular signals provides the underpinning for all neural circuitry. The initial specification of axonal and dendritic identity occurs early in neuronal life, persists for decades, and is manifested by the presence of very different sets of cell surface proteins. Yet, how neuronal polarity is established, how distinct axonal and somatodendritic domains are maintained, and how integral membrane proteins are directed to dendrites or accumulate in axons remain enduring and formidable questions in neuronal cell biology.