Analysis of Contractility and Invasion Potential of Two Canine Mammary Tumor Cell Lines

Peer reviewe

The nuclear import mechanism of SRF co-activator MKL1

An essential transcription factor Serum response factor (SRF) and its co-activators, Myocardin related transcription factors (MRTFs) regulate the expression of many target genes required for normal growth and actin cytoskeleton regulation. MKL1 (also known as MRTF-A and MAL) is one family member of MRTFs and mediates the signals from the cytoplasm to the nuclear SRF in response to changes in actin dynamics. Although it is well established that actin regulates nucleo-cytoplasmic shuttling of MKL1, the molecular mechanism of this regulation has not been characterized. Therefore the aim of this thesis was to reveal the mechanisms of MKL1 nuclear import. RNA interference (RNAi) screen identified two proteins as putative proteins mediating MKL1 nuclear localization: Importin-β (Ipoβ), which is the main import receptor in cells, and mRNA export factor Ddx19. The main purpose of this study was to confirm the hits from the RNAi screen and assess their specificity in regulating MKL1 localization. This work revealed that both Ipoβ and Ddx19 are specific and necessary factors for MKL1 nuclear import and thus required for SRF activation. We show that Ipoβ together with its adaptor protein Importin-α (Ipoα), binds to a bipartite nuclear localization signal (NLS) of MKL1, which is located in the actin-binding RPEL repeat and composed of two basic elements. Furthermore, the biochemical assays demonstrate that actin competes with Ipoα/Ipoβ heterodimer for access to the MKL1 NLS, thus explaining the inhibitory effect that actin binding has on MKL1 nuclear localization. By using advanced microscopy techniques, we show that Ddx19 adds an additional regulatory step for MKL1 nuclear import by modulating the conformation of MKL1, which affects its interaction with Ipoβ for efficient nuclear import. The ATPase cycle of Ddx19, which is crucial for its role in mRNA export, is not required in MKL1 nuclear import. In contrast, the RNA-binding activity of Ddx19 seems to be required. My work thus proposes a novel role for Ddx19, a well-known mRNA export factor and regulator of translation, in nuclear import of MKL1. In addition to MKL1, the conserved actin-binding RPEL repeat is also present in the Phosphatase and actin-regulating proteins (Phactrs). Our work demonstrates that the RPEL repeat of Phactr4 does not determine its localization in cells, but instead facilitates the competitive binding of monomeric actin and Protein phosphatase 1 (PPI) to Phactr4. This mechanism is required to control the phosphorylation status of cofilin, one of the downstream targets of PPI. Upon decrease in the cellular G-actin levels, Phactr4 activates cofilin through its binding to PPI, which leads to increase in the cellular levels of monomeric actin. Therefore our data pointed to an important role for Phactr4 in a feedback system, where actin monomers can locally regulate their own abundance. Thus this work highlights the role of RPEL repeat as a universal actin-binding site, which regulates actin homeostasis in cells.Seerumi responsiivinen faktori (SRF) on välttämätön transkriptiotekijä, joka yhdessä ko-aktivaattoreidensa kanssa säätelee monia solun normaaliin kasvuun sekä aktiinitukirangan säätelyyn tarvittavien proteiinien ilmentymistä. MKL1 (tunnetaan myös nimellä MRTF-A tai MAL) on monomeerista aktiinia (G-aktiini) sitova SRF:n koaktivaattori. Vasteena aktiinin polymerisaatiolle MKL1 kulkeutuu solun sytoplasmasta tumaan, jossa se yhdessä SRF:n kanssa aktivoi kohdegeenien transkriptiota. Aktiini on tärkeässä roolissa MKL1:n lokalisaation ja aktiivisuuden säätelijänä. MKL1 on suhteellisen suuri proteiini, joten sen kulkeutuminen tumaan täytyy tapahtua aktiivisesti. Vielä ei kuitenkaan tiedetä, miten ja mitkä proteiinin huolehtivat MKL1:n kuljetuksesta tumaan. Tämän asian selvittämiseksi seulottiin 80 tumakuljetukseen osallistuvaa proteiinia RNA-interferenssi (RNAi) menetelmää hyödyntäen. Seulonnan tuloksena löydettiin kaksi mahdollista MKL1:n lokalisaation säätelyyn osallistuvaa proteiinia: Importiini-β (Ipo-β), joka on hyvin yleinen kuljetusproteiini soluissa, sekä RNA-helikaasi Ddx19, joka osallistuu lähetti-RNA:n kuljetukseen ulos tumasta. Tämän työn pääasiallisena tarkoituksena oli varmistaa näiden proteiinien osallisuus MKL1:n lokalisaation säätelyssä. Tuloksemme osoittavat, että molemmat proteiinit, Ipo-β ja Ddx19, ovat tarpeellisia ja spesifisiä proteiineja MKL1:n kuljetuksessa tumaan ja näin ollen myös tärkeitä SRF:n aktiivisuuden säätelijöitä. Ipo-β yhdessä adaptoriproteiininsa Importiini-α:n (Ipo-α) kanssa tunnistaa MKL1:n N-terminaalisessa RPEL toistojaksossa sijaitsevan kaksiosaisen tumalokalisaatiosignaalin ja sitoutuu tähän. RPEL toistojakso voi sitoa myös aktiinia ja biokemialliset kokeemme osoittavatkin, että aktiini kilpailee Ipo-α/Ipo-β-heterodimeerin kanssa sitoutumisesta MKL1:n RPEL toistojaksoon. Hyödyntämällä edistyneitä mikroskooppitekniikoita, osoitamme, että Ddx19 osallistuu MKL1:n tumakuljetukseen muuntamalla MKL1:n konformaatiota, joka vaikuttaa MKL1:n ja Ipo-β:n väliseen interaktioon ja näin ollen tumakuljetuksen tehokkuuteen. Näytämme myös että lähetti-RNA:n kuljetuksessa tarvittava Ddx19:n ATPaasi aktiivisuus ei ole tarpeellinen MKL1:n tumakuljetuksessa. Sen sijaan Ddx19:n ominaisuus sitoa RNA:ta näyttäisi olevan tärkeää. Työni on siis tunnistanut aivan uuden roolin aiemmin lähetti-RNA:n kuljetukseen ja translaation säätelyyn liitetylle Ddx19:lle. MKL1:n lisäksi konservoitunut RPEL toistojakso esiintyy myös Phactr proteiini-perheen jäsenillä. Tuloksemme osoittavat, että toisin kuin MKL1:llä, RPEL toistojakso ei vaikuta Phactr4:n lokalisaatioon soluissa. Phactr4:n RPEL toistojakso voi sitoa sekä aktiinia että proteiini-fosfataasi I:tä (PPI) ja näiden proteiinien kilpaileva sitoutuminen Phactr4:n säätelee kofiliinin fosforylaatiota, joka on eräs PPI:n substraateista. Kun G-aktiinin konsentraatio soluissa on matala, Phactr4 aktivoi kofiliinin sitoutumalla PPI:n, mikä johtaa G-aktiinin konsentraation nousuun. Näin ollen tuloksemme osoittavat tärkeän roolin Phactr4:lle aktiinimonomeerien paikallisena säätelijänä. Tämä työ myös osoittaa että RPEL toistojakso sitoo yleisesti aktiinia ja säätelee aktiinifilamenttien ja monomeerien välistä tasapainoa soluissa

An actin-regulated importin alpha/beta-dependent extended bipartite NLS directs nuclear import of MRTF-A

Peer reviewe

Assembly of Peripheral Actomyosin Bundles in Epithelial Cells Is Dependent on the CaMKK2/AMPK Pathway

Summary Defects in the maintenance of intercellular junctions are associated with loss of epithelial barrier function and consequent pathological conditions, including invasive cancers. Epithelial integrity is dependent on actomyosin bundles at adherens junctions, but the origin of these junctional bundles is incompletely understood. Here we show that peripheral actomyosin bundles can be generated from a specific actin stress fiber subtype, transverse arcs, through their lateral fusion at cell-cell contacts. Importantly, we find that assembly and maintenance of peripheral actomyosin bundles are dependent on the mechanosensitive CaMKK2/AMPK signaling pathway and that inhibition of this route leads to disruption of tension-maintaining actomyosin bundles and re-growth of stress fiber precursors. This results in redistribution of cellular forces, defects in monolayer integrity, and loss of epithelial identity. These data provide evidence that the mechanosensitive CaMKK2/AMPK pathway is critical for the maintenance of peripheral actomyosin bundles and thus dictates cell-cell junctions through cellular force distribution.Peer reviewe

Cytokeratin 5 determines maturation of the mammary myoepithelium

At invasion, transformed mammary epithelial cells expand into the stroma through a disrupted myoepithelial (ME) cell layer and basement membrane (BM). The intact ME cell layer has thus been suggested to act as a barrier against invasion. Here, we investigate the mechanisms behind the disruption of ME cell layer. We show that the expression of basal/ME proteins CK5, CK14, and alpha-SMA altered along increasing grade of malignancy, and their loss affected the maintenance of organotypic 3D mammary architecture. Furthermore, our data suggests that loss of CK5 prior to invasive stage causes decreased levels of Zinc finger protein SNAI2 (SLUG), a key regulator of the mammary epithelial cell lineage determination. Consequently, a differentiation bias toward luminal epithelial cell type was detected with loss of mature, alpha-SMA-expressing ME cells and reduced deposition of basement membrane protein laminin-5. Therefore, our data discloses the central role of CK5 in mammary epithelial differentiation and maintenance of normal ME layer.Peer reviewe

RNA export factor Ddx19 is required for nuclear import of the SRF coactivator MKL1

Controlled transport of macromolecules between the cytoplasm and nucleus is essential for homeostatic regulation of cellular functions. For instance, gene expression entails coordinated nuclear import of transcriptional regulators to activate transcription and nuclear export of the resulting messenger RNAs for cytoplasmic translation. Here we link these two processes by reporting a novel role for the mRNA export factor Ddx19/Dbp5 in nuclear import of MKL1, the signal-responsive transcriptional activator of SRF. We show that Ddx19 is not a general nuclear import factor, and that its specific effect on MKL1 nuclear import is separate from its role in mRNA export. Both helicase and nuclear pore-binding activities of Ddx19 are dispensable for MKL1 nuclear import, but RNA binding is required. Mechanistically, Ddx19 operates by modulating the conformation of MKL1, which affects its interaction with Importin-beta for efficient nuclear import. Thus, Ddx19 participates in mRNA export, translation and nuclear import of a key transcriptional regulator.Peer reviewe

Phosphorylation acts positively and negatively to regulate MRTF-A subcellular localisation and activity

The myocardin-related transcription factors (MRTF-A and MRTF-B) regulate cytoskeletal genes through their partner transcription factor SRF. The MRTFs bind G-actin, and signal-regulated changes in cellular G-actin concentration control their nuclear accumulation. The MRTFs also undergo Rho- and ERK-dependent phosphorylation, but the function of MRTF phosphorylation, and the elements and signals involved in MRTF-A nuclear export are largely unexplored. We show that Rho-dependent MRTF-A phosphorylation reflects relief from an inhibitory function of nuclear actin. We map multiple sites of serum-induced phosphorylation, most of which are S/T-P motifs and show that S/T-P phosphorylation is required for transcriptional activation. ERK-mediated S98 phosphorylation inhibits assembly of G-actin complexes on the MRTF-A regulatory RPEL domain, promoting nuclear import. In contrast, S33 phosphorylation potentiates the activity of an autonomous Crm1-dependent N-terminal NES, which cooperates with five other NES elements to exclude MRTF-A from the nucleus. Phosphorylation thus plays positive and negative roles in the regulation of MRTF-A

Generation of stress fibers through myosin-driven reorganization of the actin cortex

Contractile actomyosin bundles, stress fibers, govern key cellular processes including migration, adhesion, and mechanosensing. Stress fibers are thus critical for developmental morphogenesis. The most prominent actomyosin bundles, ventral stress fibers, are generated through coalescence of pre-existing stress fiber precursors. However, whether stress fibers can assemble through other mechanisms has remained elusive. We report that stress fibers can also form without requirement of pre-existing actomyosin bundles. These structures, which we named cortical stress fibers, are embedded in the cell cortex and assemble preferentially underneath the nucleus. In this process, non-muscle myosin II pulses orchestrate the reorganization of cortical actin meshwork into regular bundles, which promote reinforcement of nascent focal adhesions, and subsequent stabilization of the cortical stress fibers. These results identify a new mechanism by which stress fibers can be generated de novo from the actin cortex and establish role for stochastic myosin pulses in the assembly of functional actomyosin bundles.Peer reviewe

Mechanosensitive TRPV4 channel guides maturation and organization of the bilayered mammary epithelium

Abstract Biophysical cues from the cell microenvironment are detected by mechanosensitive components at the cell surface. Such machineries convert physical information into biochemical signaling cascades within cells, subsequently leading to various cellular responses in a stimulus-dependent manner. At the surface of extracellular environment and cell cytoplasm exist several ion channel families that are activated by mechanical signals to direct intracellular events. One of such channel is formed by transient receptor potential cation channel subfamily V member, TRPV4 that is known to act as a mechanosensor in wide variaty of tissues and control ion-influx in a spatio-temporal way. Here we report that TRPV4 is prominently expressed in the stem/progenitor cell populations of the mammary epithelium and seems important for the lineage-specific differentiation, consequently affecting mechanical features of the mature mammary epithelium. This was evident by the lack of several markers for mature myoepithelial and luminal epithelial cells in TRPV4-depleted cell lines. Interestingly, TRPV4 expression is controlled in a tension-dependent manner and it also impacts differentation process dependently on the stiffness of the microenvironment. Furthermore, such cells in a 3D compartment were disabled to maintain normal mammosphere structures and displayed abnormal lumen formation, size of the structures and disrupted cellular junctions. Mechanosensitive TRPV4 channel therefore act as critical player in the homeostasis of normal mammary epithelium through sensing the physical environment and guiding accordingly differentiation and structural organization of the bilayered mammary epithelium