Involvement of non-muscle α-actinins and NUAK2 kinase in regulating actin stress fibers

Actin cytoskeleton is essential in generating mechanical forces together with the associated adhesions and transmitting signals that impact processes such as cell migration. Cell migration is necessary for numerous biological processes including wound healing and embryonic development. Moreover, aberrant cell migration promotes cancer invasion and metastasis. Cell migration events require dramatic spatial and temporal reorganization of the actin cytoskeleton that involves coordinated formation and regulation of multiple structures such as actin stress fibers. Actin stress fibers are dynamic structures, which differ in their subcellular localization, connection to substratum and their dynamics. However, these actin stress fibers are less characterized in terms of the molecules required for assembly-disassembly and the signaling pathways involved in regulating their functions in mesenchymal and epithelial cells. This thesis focuses on characterizing key players and signaling pathways involved in regulating actin stress fiber assembly-disassembly, cell adhesion and contractility. Understanding these cell plasticity changes is essential as in cancer context they are likely to be deregulated thus leading to increased migratory and invasive potential of the cells. During this thesis study, NUAK2 a novel serine-threonine kinase was identified to associate with myosin phosphatase Rho-interacting protein (MRIP) on actin stress fibers. Association between NUAK2 and MRIP increases cells contractility and promotes formation of actin stress fibers through phosphorylation of myosin light chain (MLC). The identified NUAK2-MRIP association reveals a novel mechanism for the maintenance of actin stress fibers. Our findings implicate NUAK2 as an important regulator of cell contractility and actin stress fiber assembly. Thus providing further knowledge of how actin stress fibers and cell contractility can be regulated in mesenchymal cells. To further characterize the specificity of molecules required for the assembly of actin stress fibers, we studied the function of most abundant actin crosslinking proteins in non-muscle cells, α-actinin-1 and α-actinin-4. Our findings reveal that specifically α-actinin-1 and not α-actinin-4, is required to assemble dorsal stress fibers found at the leading edge of mesenchymal cells. In addition, loss of α-actinin-1 modulates cell-matrix adhesions leading to decreased cell migration without altering cells contractility. Contrary to traditional views, dorsal stress fibers assembled by α-actinin-1 are non-contractile and are induced by Rac1 signaling. Rac1 is essential in regulating polymerization of actin filaments. Thus suggesting that force required for cell migration is at least partially generated through actin polymerization. Interestingly, we found α-actinin-1 to be upregulated in various cancers and especially associates with decreased survival in estrogen receptor (ER) negative breast cancer patients. In mammary epithelial cells, α-actinin-1 levels regulate epithelial cell plasticity, reorganize actin stress fibers and destabilize cell-cell adhesions accompanied with increased cell migration. This thesis extends the knowledge of especially α-actinin-1 in regulating actin stress fiber assembly and cell plasticity in both epithelial and mesenchymal cells. Furthermore, identifying α-actinin-1 as a candidate prognostic biomarker in ER negative breast cancer patients.Solujen kyky liikkua, muuttaa muotoaan ja muokkautua ympäröivään kudokseen sekä mekaanisiin ärsykkeisiin ovat keskeisessä asemassa lukuisissa biologisissa prosesseissa kuten haavan paranemisessa ja alkionkehityksessä. Patologisissa olosuhteissa kuten syövässä solut liikkuvat hallitsemattomasti, mikä puolestaan johtaa niiden kudosrajoista piittaamattomaan leviämiseen ympäröivään kudokseen ja sitä kautta muihin elimiin. Liikkuakseen solu tarvitsee aktiinitukirankaa, joka tuottaa liikkumiseen tarvittavaa voimaa erityisesti supistuvien aktiinisäikeiden avulla. Aktiinisäikeet voivat sitoutua solun kasvupohjassa sijaitseviin reseptoreihin ja siten säädellä solujen kiinnittymistä ympäröivään kudokseen sekä aikaansaada voimia, joiden avulla solu kykenee liikkumaan hallitusti eteenpäin. Tämän väitöskirjatyön päämääränä oli tutkia liikkumisen kannalta keskeisten aktiinisäikeiden molekulaarinen koostumus ja säätelymekanismit sekä niiden merkitys syöpäsolujen liikkumisessa ja vuorovaikutuksessa. Väitöskirjatyön aikana tunnistettiin uusi vuorovaikutus aiemmin tunnetun aktiinisäieproteiini MRIP:n sekä NUAK2-kinaasin välillä. Tämä vuorovaikutus paljasti uuden mekanismin, jonka avulla solut voivat säädellä aktiinisäikeiden muodostumista, sekä niiden supistumista. Lisäksi havaittiin, että NUAK2-kinaasin ilmentymistasot voi säädellä supistuvien aktiinisäikeiden määrää. Nämä havainnot ovat tärkeitä ja ne lisäävät merkittävästi tietämystä siitä, minkälaisilla mekanismeilla aktiinisäikeitä voidaan säädellä. Selvittääksemme tarkemmin aktiinisäikeiden molekulaarista koostumusta, tutkimme väitöskirjatyössäni myös aktiinisäikeitä niputtavia proteiineja α-aktiniini-1 ja α-aktiniini-4. Tutkimuksemme osoitti, että erityisesti α-aktiniini-1 on välttämätön ns. dorsaalisten aktiinisäikeiden niputtaja ja, että nämä säikeet edistävät solujen liikkumista. Tutkimuksemme paljastivat myös, että α-aktiniini-1 on erittäin keskeisessä asemassa mesenkymaalisten solujen kiinnittymisessä kasvupohjan reseptoreihin. Lisäksi havaitsimme rintarauhasen epiteelisoluissa, että α-aktiniini-1:n ilmentymistasot säätelevät aktiinisäikeiden uudelleenjärjestäytymistä, solujen välistä yhteyttä, sekä solujen liikkumista. Löydöksemme osoittavat myös, että α-aktiniini-1:n ilmentymistaso on lisääntynyt useissa syövissä, ja tämän on havaittu olevan sidoksissa heikentyneeseen eloonjäämisennusteeseen erityisesti estrogeenireseptorin (ER) suhteen negatiivisilla rintasyöpäpotilailla. Tämän väitöskirjatyön tulokset laajentavat erityisesti tietämystä α-aktiniini-1:n roolista aktiinisäikeiden säätelijänä ja solujen plastisuuden muokkaajana niin mesenkymaalisissa kuin epiteelisoluissakin. Tutkimustuloksemme viittaavat siihen, että α-aktiniini-1 voisi toimia yhtenä mahdollisena prognostisena biomarkerina ER:n suhteen negatiivisilla rintasyöpäpotilailla. Väitöskirjatyöni tutkimustulokset selvittävät aktiinisäikeiden sekä niiden säätelijöiden kuten, α-aktiniini-1, merkitystä syöpäsolujen vuorovaikutuksessa ja niiden hallitsemattomassa liikkumisessa. Parhaimmillaan tämä tieto voi edesauttaa syövän diagnostiikkaa ja hoitomenetelmien kehittämistä

Role and function of nonmuscle alpha-actinin-1 and -4 in regulating distinct subcategories of actin stress fibers in mammalian cells

Actin stress fibers are dynamic structures in the cytoskeleton, which respond to mechanical stimuli and affect cell motility, adhesion and invasion of cancer cells. In nonmuscle cells, stress fibers have been subcategorized to three distinct stress fiber types: dorsal and ventral stress fibers and transverse arcs. These stress fibers are dissimilar in their subcellular localization, connection to substratum as well as in their dynamics and assembly mechanisms. Still uncharacterized is how they differ in their function and molecular composition. Here, I have studied involvement of nonmuscle alpha-actinin-1 and -4 in regulating distinct stress fibers as well as their localization and function in human U2OS osteosarcoma cells. Except for the correlation of upregulation of alpha-actinin-4 in invasive cancer types very little is known about whether these two actinins are redundant or have specific roles. The availability of highly specific alpha-actinin-1 antibody generated in the lab, revealed localization of alpha-actinin-1 along all three categories of stress fibers while alphaactinin-4 was detected at cell edge, distal ends of stress fibers as well as perinuclear regions. Strikingly, by utilizing RNAi-mediated gene silencing of alpha-actinin-1 resulted in specific loss of dorsal stress fibers and relocalization of alpha-actinin-4 to remaining transverse arcs and ventral stress fibers. Unexpectedly, aberrant migration was not detected in cells lacking alpha-actinin-1 even though focal adhesions were significantly smaller and fewer. Whereas, silencing of alpha-actinin-4 noticeably affected overall cell migration. In summary, as part of my master thesis study I have been able to demonstrate distinct localization and functional patterns for both alpha-actinin-1 and -4. I have identified alpha-actinin-1 to be a selective dorsal stress fiber crosslinking protein as well as to be required for focal adhesion maturation, while alpha-actinin-4 was demonstrated to be fundamental for cell migration

Increased alpha-actinin-1 destabilizes E-cadherin-based adhesions and associates with poor prognosis in basal-like breast cancer

The controlled formation and stabilization of E-cadherin-based adhesions is vital for epithelial integrity. This requires co-operation between the E-cadherin-based adhesions and the associated actin cytoskeleton. In cancer, this co-operation often fails, predisposing cells to migration through molecular mechanisms that have only been partially characterized. Here, we demonstrate that the actin filament cross-linker alpha-actinin-1 is frequently increased in human breast cancer. In mammary epithelial cells, the increased alpha-actinin-1 levels promote cell migration and induce disorganized acini-like structures in Matrigel. This is accompanied by a major reorganization of the actin cytoskeleton and the associated E-cadherin-based adhesions. Increased expression of alpha-actinin-1 is particularly noted in basal-like breast cancer cell lines, and in breast cancer patients it associates with poor prognosis in basal-like subtypes. Downregulation of alpha-actinin-1 in E-cadherin expressing basal-like breast cancer cells demonstrate that alpha-actinin-1-assembled actin fibers destabilize E-cadherin-based adhesions. Taken together, these results indicate that increased alpha-actinin-1 expression destabilizes E-cadherin-based adhesions, which is likely to promote the migratory potential of breast cancer cells. Furthermore, our results identify alpha-actinin-1 as a candidate prognostic biomarker in basal-like breast cancer.Peer reviewe

The evolution of the density of galaxy clusters and groups: denser environments at higher redshifts

We show that, observationally, the projected local density distribution in high-z clusters is shifted towards higher values compared to clusters at lower redshift. To search for the origin of this evolution, we analyze a sample of haloes selected from the Millennium Simulation and populated using semi-analytic models, investigating the relation between observed projected density and physical 3D density, using densities computed from the 10 and 3 closest neighbours. Both observationally and in the simulations, we study the relation between number of cluster members and cluster mass, and number of members per unit of cluster mass. We find that the observed evolution of projected densities reflects a shift to higher values of the physical 3D density distribution. In turn, this must be related with the globally higher number of galaxies per unit of cluster volume N/V in the past. We show that the evolution of N/V is due to a combination of two effects: a) distant clusters were denser in dark matter (DM) simply because the DM density within R_{200} (~the cluster virial radius) is defined to be a fixed multiple of the critical density of the Universe, and b) the number of galaxies per unit of cluster DM mass is remarkably constant both with redshift and cluster mass if counting galaxies brighter than a passively evolving magnitude limit. Our results highlight that distant clusters were much denser environments than today's clusters, both in galaxy number and mass, and that the density conditions felt by galaxies in virialized systems do not depend on the system mass.Comment: accepted for publication in MNRA

Galaxy stellar mass functions of different morphological types in clusters, and their evolution between z=0.8 and z=0

We present the galaxy stellar mass function (MF) and its evolution in clusters from z~0.8 to the current epoch, based on the WIde-field Nearby Galaxy-cluster Survey (WINGS) (0.04<z<0.07), and the ESO Distant Cluster Survey (EDisCS) (0.4<z <0.8). We investigate the total MF and find it evolves noticeably with redshift. The shape at M*>10^11 M' does not evolve, but below M*~10^10.8 M' the MF at high redshift is flat, while in the Local Universe it flattens out at lower masses. The population of M* = 10^10.2 - 10^10.8 M' galaxies must have grown significantly between z=0.8 and z=0. We analyze the MF of different morphological types (ellipticals, S0s and late-types), and find that also each of them evolves with redshift. All types have proportionally more massive galaxies at high- than at low-z, and the strongest evolution occurs among S0 galaxies. Examining the morphology-mass relation (the way the proportion of galaxies of different morphological types changes with galaxy mass), we find it strongly depends on redshift. At both redshifts, ~40% of the stellar mass is in elliptical galaxies. Another ~43% of the mass is in S0 galaxies in local clusters, while it is in spirals in distant clusters. To explain the observed trends, we discuss the importance of those mechanisms that could shape the MF. We conclude that mass growth due to star formation plays a crucial role in driving the evolution. It has to be accompanied by infall of galaxies onto clusters, and the mass distribution of infalling galaxies might be different from that of cluster galaxies. However, comparing with high-z field samples, we do not find conclusive evidence for such an environmental mass segregation. Our results suggest that star formation and infall change directly the MF of late-type galaxies in clusters and, indirectly, that of early-type galaxies through subsequent morphological transformations.Comment: MNRAS in press, 24 pages, 19 figures and 8 table

NETosis in Alzheimer's Disease

Alzheimer's disease (AD) is a neurodegenerative disorder characterized by the progressive deterioration of cognitive functions. Its neuropathological features include amyloid-\u3b2 (A\u3b2) accumulation, the formation of neurofibrillary tangles, and the loss of neurons and synapses. Neuroinflammation is a well-established feature of AD pathogenesis, and a better understanding of its mechanisms could facilitate the development of new therapeutic approaches. Recent studies in transgenic mouse models of AD have shown that neutrophils adhere to blood vessels and migrate inside the parenchyma. Moreover, studies in human AD subjects have also shown that neutrophils adhere and spread inside brain vessels and invade the parenchyma, suggesting these cells play a role in AD pathogenesis. Indeed, neutrophil depletion and the therapeutic inhibition of neutrophil trafficking, achieved by blocking LFA-1 integrin in AD mouse models, significantly reduced memory loss and the neuropathological features of AD. We observed that neutrophils release neutrophil extracellular traps (NETs) inside blood vessels and in the parenchyma of AD mice, potentially harming the blood-brain barrier and neural cells. Furthermore, confocal microscopy confirmed the presence of NETs inside the cortical vessels and parenchyma of subjects with AD, providing more evidence that neutrophils and NETs play a role in AD-related tissue destruction. The discovery of NETs inside the AD brain suggests that these formations may exacerbate neuro-inflammatory processes, promoting vascular and parenchymal damage during AD. The inhibition of NET formation has achieved therapeutic benefits in several models of chronic inflammatory diseases, including autoimmune diseases affecting the brain. Therefore, the targeting of NETs may delay AD pathogenesis and offer a novel approach for the treatment of this increasingly prevalent disease

VizieR Online Data Catalog: EELGs out to z~1 in zCOSMOS (Amorin+, 2015)

Star formation rates have been computed from Hα or Hβ luminosity (IMF from Chabrier et al. (2003PASP..115..763C) and assuming a theoretical ratio Hα/Hβ=2.82) following Kennicutt (1998ApJ...498..541K). Uncertainties in SFR account for the propagation of errors in line fluxes and reddening. Gas-phase metallicity has been derived using four methods: (1) the direct method (Hagele et al., 2008MNRAS.383..209H); (2) the Te-Z correlation (This work); (3) the N2 calibration (Perez-Montero & Contini, 2009MNRAS.398..949P); and (4) the R23 calibration (McGaugh, 1991ApJ...380..140M) scaled to the direct method using the linear relation presented by Lamareille et al. (2006, Cat. J/A+A/448/893 and 2006A&A...448..907L, see also Perez-Montero et al., 2013A&A...549A..25P). In all cases, 1σ uncertainties in metallicity account for the propagated errors in line fluxes and reddening. (2 data files)

Search for new particles in events with energetic jets and large missing transverse momentum in proton-proton collisions at root s=13 TeV

A search is presented for new particles produced at the LHC in proton-proton collisions at root s = 13 TeV, using events with energetic jets and large missing transverse momentum. The analysis is based on a data sample corresponding to an integrated luminosity of 101 fb(-1), collected in 2017-2018 with the CMS detector. Machine learning techniques are used to define separate categories for events with narrow jets from initial-state radiation and events with large-radius jets consistent with a hadronic decay of a W or Z boson. A statistical combination is made with an earlier search based on a data sample of 36 fb(-1), collected in 2016. No significant excess of events is observed with respect to the standard model background expectation determined from control samples in data. The results are interpreted in terms of limits on the branching fraction of an invisible decay of the Higgs boson, as well as constraints on simplified models of dark matter, on first-generation scalar leptoquarks decaying to quarks and neutrinos, and on models with large extra dimensions. Several of the new limits, specifically for spin-1 dark matter mediators, pseudoscalar mediators, colored mediators, and leptoquarks, are the most restrictive to date.Peer reviewe

Probing effective field theory operators in the associated production of top quarks with a Z boson in multilepton final states at root s=13 TeV

Peer reviewe

Assembly of non-contractile dorsal stress fibers requires α-actinin-1 and Rac1 in migrating and spreading cells

Cell migration and spreading is driven by actin polymerization and actin stress fibers. Actin stress fibers are considered to contain aactinin crosslinkers and nonmuscle myosin II motors. Although several actin stress fiber subtypes have been identified in migrating and spreading cells, the degree of molecular diversity of their composition and the signaling pathways regulating fiber subtypes remain largely uncharacterized. In the present study we identify that dorsal stress fiber assembly requires α-actinin-1. Loss of dorsal stress fibers in α-actinin-1-depleted cells results in defective maturation of leading edge focal adhesions. This is accompanied by a delay in early cell spreading and slower cell migration without noticeable alterations in myosin light chain phosphorylation. In agreement with the unaltered myosin II activity, dorsal stress fiber trunks lack myosin II and are resistant to myosin II ATPase inhibition. Furthermore, the non-contractility of dorsal stress fibers is supported by the finding that Rac1 induces dorsal stress fiber assembly whereas contractile ventral stress fibers are induced by RhoA. Loss of dorsal stress fibers either by depleting α-actinin-1 or Rac1 results in a β-actin accumulation at the leading edge in migrating and spreading cells. These findings molecularly specify dorsal stress fibers from other actin stress fiber subtypes. Furthermore, we propose that non-contractile dorsal stress fibers promote cell migration and early cell spreading through Rac1-induced actin polymerization