Susy QCD and High Energy Cosmic Rays 1. Fragmentation functions of Susy QCD

The supersymmetric evolution of the fragmentation functions (or timelike evolution) within N=1 $QCD$ is discussed and predictions for the fragmentation functions of the theory (into final protons) are given. We use a backward running of the supersymmetric DGLAP equations, using a method developed in previous works. We start from the usual QCD parameterizations at low energy and run the DGLAP back, up to an intermediate scale -assumed to be supersymmetric- where we switch-on supersymmetry. From there on we assume the applicability of an N=1 supersymmetric evolution (ESAP). We elaborate on possible application of these results to High Energy Cosmic Rays near the GZK cutoff.Comment: 36 pages, 12 fig

Functional characterisation of a novel microtubule-actin interacting protein, GAS2-like 3

The microtubule (MT) and actin cytoskeletons are fundamental to cell integrity, as they control a host of cellular activities. Until recently, studies have investigated these cytoskeletal components as separate entities, however, it has become increasingly clear that the MT and actin cytoskeletons function in an interdependent way. Elucidating how the two components interact will be key to our further understanding of fundamental cellular processes such as cell division, growth, polarisation and migration. This study focused on a newly identified and highly conserved protein, named GAS2-like 3, which contains a putative actin binding calponin homology (CH) domain and a putative MT binding GAS2-related (GAR) domain. GAS2-like 3 predominantly localised to both MT and actin cytoskeletons. By using a series of truncation mutants, the fundamental regions of GAS2-like 3 involved in mediating these interactions were dissected, both in cells and in vitro. Data from sedimentation assays revealed the CH domain binds directly to F-actin, and the C-terminus binds directly to MTs, whereas the GAR domain does not interact directly with MTs. In order to assess the dynamics of GAS2-like 3 and its mutants in cells, Fluorescence Recovery After Photobleaching experiments were performed to reveal that the GAR domain of GAS2-like 3 modulates the binding strength of GAS2-like 3 to MTs and actin. GAS2-like 3 localisation is dependent on both MT and actin cytoskeletons, although MTs have a greater influence on GAS2-like 3 localisation. Interestingly, tubulin acetylation enhanced GAS2-like 3 localisation to MTs. This study also uncovered two binding motifs for the MT plus-end binding protein 1 (EB1). EB1 is characterised as a new interaction partner of GAS2-like 3, which directly interacts with the C-terminus of GAS2-like 3 in vitro. Furthermore, a molecular mechanism in which GAS2-like 3 is recruited to MTs via EB1 in cells is revealed. Interestingly, the interaction between EB1 and GAS2-like 3 requires the complete C-terminus, rather than the specific EB1-binding motifs themselves.Taken together, this study provides insights into the new protein, GAS2-like 3, which has the capacity to crosslink MTs and actin and provides a mechanistic insight into how GAS2-like 3 localisation is regulated in cells.EThOS - Electronic Theses Online ServiceGBUnited Kingdo

Marching at the front and dragging behind: differential αVβ3-integrin turnover regulates focal adhesion behavior

Integrins are cell–substrate adhesion molecules that provide the essential link between the actin cytoskeleton and the extracellular matrix during cell migration. We have analyzed αVβ3-integrin dynamics in migrating cells using a green fluorescent protein–tagged β3-integrin chain. At the cell front, adhesion sites containing αVβ3-integrin remain stationary, whereas at the rear of the cell they slide inward. The integrin fluorescence intensity within these different focal adhesions, and hence the relative integrin density, is directly related to their mobility. Integrin density is as much as threefold higher in sliding compared with stationary focal adhesions. High intracellular tension under the control of RhoA induced the formation of high-density contacts. Low-density adhesion sites were induced by Rac1 and low intracellular tension. Photobleaching experiments demonstrated a slow turnover of β3-integrins in low-density contacts, which may account for their stationary nature. In contrast, the fast β3-integrin turnover observed in high-density contacts suggests that their apparent sliding may be caused by a polarized renewal of focal contacts. Therefore, differential acto-myosin–dependent integrin turnover and focal adhesion densities may explain the mechanical and behavioral differences between cell adhesion sites formed at the front, and those that move in the retracting rear of migrating cells

Vinculin controls focal adhesion formation by direct interactions with talin and actin

Focal adhesions (FAs) regulate cell migration. Vinculin, with its many potential binding partners, can interconnect signals in FAs. Despite the well-characterized structure of vinculin, the molecular mechanisms underlying its action have remained unclear. Here, using vinculin mutants, we separate the vinculin head and tail regions into distinct functional domains. We show that the vinculin head regulates integrin dynamics and clustering and the tail regulates the link to the mechanotransduction force machinery. The expression of vinculin constructs with unmasked binding sites in the head and tail regions induces dramatic FA growth, which is mediated by their direct interaction with talin. This interaction leads to clustering of activated integrin and an increase in integrin residency time in FAs. Surprisingly, paxillin recruitment, induced by active vinculin constructs, occurs independently of its potential binding site in the vinculin tail. The vinculin tail, however, is responsible for the functional link of FAs to the actin cytoskeleton. We propose a new model that explains how vinculin orchestrates FAs

The structural basis of the Talin-KANK1 interaction that coordinates the actin and microtubule cytoskeletons at focal adhesions

Adhesion between cells and the extracellular matrix (ECM) is mediated by heterodimeric (alphabeta) integrin receptors that are intracellularly linked to the contractile actomyosin machinery. One of the proteins that control this link is talin, which organises cytosolic signalling proteins into discrete complexes on beta-integrin tails referred to as focal adhesions (FAs). The adapter protein KANK1 binds to talin in the region of FAs known as the adhesion belt. Here, we developed a novel crystallographic method to resolve the talin-KANK1 complex. This structure revealed that the talin binding KN motif of KANK1 has a novel fold, where a beta-turn stabilises the alpha-helical region, explaining its specific interaction with talin R7 and high affinity. Single point mutants in KANK1 identified from the structure abolished the interaction and enabled us to examine KANK1 enrichment in the adhesion belt. Strikingly, in cells expressing a constitutively active form of vinculin that keeps the FA structure intact even in the presence of myosin inhibitors, KANK1 localises throughout the entire FA structure even when actomyosin tension is released. We propose a model whereby actomyosin forces on talin eliminate KANK1 from talin binding in the centre of FAs while retaining it at the adhesion periphery

Estimation of Cell Cycle States of Human Melanoma Cells with Quantitative Phase Imaging and Deep Learning

Visualization and classification of cell cycle stages in live cells requires the introduction of transient or stably expressing fluorescent markers. This is not feasible for all cell types, and can be time consuming to implement. Labelling of living cells also has the potential to perturb normal cellular function. Here we describe a computational strategy to estimate core cell cycle stages without markers by taking advantage of features extracted from information-rich ptychographic time-lapse movies. We show that a deep-learning approach can estimate the cell cycle trajectories of individual human melanoma cells from short 3-frame (~23 minute) snapshots, and can identify cell cycle arrest induced by chemotherapeutic agents targeting melanoma driver mutations

Modulation of FAK and Src adhesion signaling occurs independently of adhesion complex composition

Integrin adhesion complexes (IACs) form mechanochemical connections between the extracellular matrix and actin cytoskeleton and mediate phenotypic responses via posttranslational modifications. Here, we investigate the modularity and robustness of the IAC network to pharmacological perturbation of the key IAC signaling components focal adhesion kinase (FAK) and Src. FAK inhibition using AZ13256675 blocked FAK(Y397) phosphorylation but did not alter IAC composition, as reported by mass spectrometry. IAC composition was also insensitive to Src inhibition using AZD0530 alone or in combination with FAK inhibition. In contrast, kinase inhibition substantially reduced phosphorylation within IACs, cell migration and proliferation. Furthermore using fluorescence recovery after photobleaching, we found that FAK inhibition increased the exchange rate of a phosphotyrosine (pY) reporter (dSH2) at IACs. These data demonstrate that kinase-dependent signal propagation through IACs is independent of gross changes in IAC composition. Together, these findings demonstrate a general separation between the composition of IACs and their ability to relay pY-dependent signals

The βI domain promotes active β1 integrin clustering into mature adhesion sites

Modulation of integrin function is required in many physiological and pathological settings, such as angiogenesis and cancer. Integrin allosteric changes, clustering, and trafficking cooperate to regulate cell adhesion and motility on extracellular matrix proteins via mechanisms that are partly defined. By exploiting four monoclonal antibodies recognizing distinct conformational epitopes, we show that in endothelial cells (ECs), the extracellular βI domain, but not the hybrid or I-EGF2 domain of active β1 integrins, promotes their FAK-regulated clustering into tensin 1–containing fibrillar adhesions and impairs their endocytosis. In this regard, the βI domain–dependent clustering of active β1 integrins is necessary to favor fibronectin-elicited directional EC motility, which cannot be effectively promoted by β1 integrin conformational activation alone

Syndecan-4 phosphorylation is a control point for integrin recycling

Precise spatiotemporal coordination of integrin adhesion complex dynamics is essential for efficient cell migration. For cells adherent to fibronectin, differential engagement of α5β1 and αVβ3 integrins is used to elicit changes in adhesion complex stability, mechanosensation, matrix assembly, and migration, but the mechanisms responsible for receptor regulation have remained largely obscure. We identify phosphorylation of the membrane-intercalated proteoglycan syndecan-4 as an essential switch controlling integrin recycling. Src phosphorylates syndecan-4 and, by driving syntenin binding, leads to suppression of Arf6 activity and recycling of αVβ3 to the plasma membrane at the expense of α5β1. The resultant elevation in αVβ3 engagement promotes stabilization of focal adhesions. Conversely, abrogation of syndecan-4 phosphorylation drives surface expression of α5β1, destabilizes adhesion complexes, and disrupts cell migration. These data identify the dynamic spatiotemporal regulation of Src-mediated syndecan-4 phosphorylation as an essential switch controlling integrin trafficking and adhesion dynamics to promote efficient cell migration

The C terminus of talin links integrins to cell cycle progression

Talin recruits and activates focal adhesion proteins required for cell cycle progression