Browsing by Subject "Actin"

Sort by: Order: Results:

Now showing items 1-12 of 12
  • Peippo, Minna; Gardberg, Maria; Lamminen, Tarja; Kaipio, Katja; Carpen, Olli; Heuser, Vanina D. (2017)
    The functional properties of actin-regulating formin proteins are diverse and in many cases cell-type specific. FHOD1, a formin expressed predominantly in cells of mesenchymal lineage, bundles actin filaments and participates in maintenance of cell shape, migration and cellular protrusions. FHOD1 participates in cancer associated epithelial to mesenchymal transition (EMT) in oral squamous cell carcinoma and breast cancer. The role of FHOD1 in melanomas has not been characterized. Here, we show that FHOD1 expression is typically strong in cutaneous melanomas and cultured melanoma cells while the expression is low or absent in benign nevi. By using shRNA to knockdown FHOD1 in melanoma cells, we discovered that FHOD1 depleted cells are larger, rounder and have smaller focal adhesions and inferior migratory capacity as compared to control cells. Importantly, we found FHOD1 depleted cells to have reduced colony-forming capacity and attenuated tumor growth in vivo, a finding best explained by the reduced proliferation rate caused by cell cycle arrest. Unexpectedly, FHOD1 depletion did not prevent invasive growth at the tumor margins. These results suggest that FHOD1 participates in key cellular processes that are dysregulated in malignancy, but may not be essential for melanoma cell invasion.
  • Ross, Jacob A.; Levy, Yotam; Ripolone, Michela; Kolb, Justin S.; Turmaine, Mark; Holt, Mark; Lindqvist, Johan; Claeys, Kristl G.; Weis, Joachim; Monforte, Mauro; Tasca, Giorgio; Moggio, Maurizio; Figeac, Nicolas; Zammit, Peter S.; Jungbluth, Heinz; Fiorillo, Chiara; Vissing, John; Witting, Nanna; Granzier, Henk; Zanoteli, Edmar; Hardeman, Edna C.; Wallgren-Pettersson, Carina; Ochala, Julien (2019)
    Nemaline myopathy (NM) is a skeletal muscle disorder caused by mutations in genes that are generally involved in muscle contraction, in particular those related to the structure and/or regulation of the thin filament. Many pathogenic aspects of this disease remain largely unclear. Here, we report novel pathological defects in skeletal muscle fibres of mouse models and patients with NM: irregular spacing and morphology of nuclei; disrupted nuclear envelope; altered chromatin arrangement; and disorganisation of the cortical cytoskeleton. Impairments in contractility are the primary cause of these nuclear defects. We also establish the role of microtubule organisation in determining nuclear morphology, a phenomenon which is likely to contribute to nuclear alterations in this disease. Our results overlap with findings in diseases caused directly by mutations in nuclear envelope or cytoskeletal proteins. Given the important role of nuclear shape and envelope in regulating gene expression, and the cytoskeleton in maintaining muscle fibre integrity, our findings are likely to explain some of the hallmarks of NM, including contractile filament disarray, altered mechanical properties and broad transcriptional alterations.
  • Raza, Shaffaq (Helsingin yliopisto, 2020)
    Growth differentiation factor 15 (GDF15), a member of TGF-β super family is a soluble cytokine that is associated with different pathological conditions including cancer, cardiac and renal failure and obesity. Its high serum levels are linked with symptoms like cachexia/anorexia in cancer patients and can be used as a marker for these diseases. Its crucial role in weight regulation and energy homeostasis has been demonstrated by treating obese mice with GDF15, which results in weight lose along with improved glucose metabolism and increased insulin tolerance. It is now known that GDF15 exerts its metabolic effect by binding to a GDNF receptor -α-Like (GFRAL) receptor along with co-receptor RET. Interestingly, these two receptors co-localize only in the brain stem area of mice and humans indicating involvement of a neuronal circuit in GDF15 mediated effects. Despite its implications in major health disorders, little is known about the interaction of GDF15 with its receptors and how this interaction in turn modulates different cellular signalling and functions. The aim of the thesis was to study the mechanism and factors involved in endocytosis of GDF15. I employed high content imaging and flow cytometry techniques to visualize and analyse the internalization of ligand-receptor complex and investigate the role of actin, dynamin and phosphoinositide 3 kinase in the process. The results suggest that similar to the internalization of other cellular growth factors, the uptake of GDF15 is affected by disruption of the actin cytoskeleton. The role of dynamin is still unclear. I also discovered that the internalization of GDF15 was inefficient even in cells that expressed the receptor GFRAL, with large cell-to-cell variation. By following the intracellular localization of the receptor GFRAL, my results revealed that the receptor GFRAL is not efficiently exported to the plasma membrane and most of the protein is retained in the Golgi compartment of cells. This phenomenon was stronger in murine fibroblast cells, where the receptor was almost exclusively trapped in the secretory compartment, explaining why the uptake of the ligand GDF15 is so inefficient in these cells. The system developed during this project will now be used to analyse different factors involved in the uptake of GDF15 and eventually uncover the possible endocytic pathway. Moreover, the Golgi retention of the receptor opens up new questions to investigate like whether the physiological function of GDF15 is regulated by receptor export signals. This will help deciphering the complex and mysterious interaction of GDF15 with its receptor GFRAL.
  • Heuser, Vanina D.; Kiviniemi, Aida; Lehtinen, Laura; Munthe, Sune; Kristensen, Bjarne Winther; Posti, Jussi P.; Sipilä, Jussi O. T.; Vuorinen, Ville; Carpen, Olli; Gardberg, Maria (2020)
    BackgroundThe prognosis of glioblastoma remains poor, related to its diffuse spread within the brain. There is an ongoing search for molecular regulators of this particularly invasive behavior. One approach is to look for actin regulating proteins that might be targeted by future anti-cancer therapy. The formin family of proteins orchestrates rearrangement of the actin cytoskeleton in multiple cellular processes. Recently, the formin proteins mDia1 and mDia2 were shown to be expressed in glioblastoma in vitro, and their function could be modified by small molecule agonists. This finding implies that the formins could be future therapeutic targets in glioblastoma.MethodsIn cell studies, we investigated the changes in expression of the 15 human formins in primary glioblastoma cells and commercially available glioblastoma cell lines during differentiation from spheroids to migrating cells using transcriptomic analysis and qRT-PCR. siRNA mediated knockdown of selected formins was performed to investigate whether their expression affects glioblastoma migration.Using immunohistochemistry, we studied the expression of two formins, FHOD1 and INF2, in tissue samples from 93 IDH-wildtype glioblastomas. Associated clinicopathological parameters and follow-up data were utilized to test whether formin expression correlates with survival or has prognostic value.ResultsWe found that multiple formins were upregulated during migration. Knockdown of individual formins mDia1, mDia2, FHOD1 and INF2 significantly reduced migration in most studied cell lines. Among the studied formins, knockdown of INF2 generated the greatest reduction in motility in vitro. Using immunohistochemistry, we demonstrated expression of formin proteins FHOD1 and INF2 in glioblastoma tissues. Importantly, we found that moderate/high expression of INF2 was associated with significantly impaired prognosis.ConclusionsFormins FHOD1 and INF2 participate in glioblastoma cell migration. Moderate/high expression of INF2 in glioblastoma tissue is associated with worse outcome. Taken together, our in vitro and tissue studies suggest a pivotal role for INF2 in glioblastoma. When specific inhibiting compounds become available, INF2 could be a target in the search for novel glioblastoma therapies.
  • Marttila, Minttu; Mubashir, Hanif; Lemola, Elina; Nowak, Kristen J.; Laitila, Jenni; Gronholm, Mikaela; Wallgren-Pettersson, Carina; Pelin, Katarina (2014)
    Background: Nemaline myopathy (NM) is a rare genetic muscle disorder, but one of the most common among the congenital myopathies. NM is caused by mutations in at least nine genes: Nebulin (NEB), alpha-actin (ACTA1), alpha-tropomyosin (TPM3), beta-tropomyosin (TPM2), troponin T (TNNT1), cofilin-2 (CFL2), Kelch repeat and BTB (POZ) domain-containing 13 (KBTBD13), and Kelch-like family members 40 and 41 (KLHL40 and KLHL41). Nebulin is a giant (600 to 900 kDa) filamentous protein constituting part of the skeletal muscle thin filament. Around 90% of the primary structure of nebulin is composed of approximately 35-residue alpha-helical domains, which form super repeats that bind actin with high affinity. Each super repeat has been proposed to harbor one tropomyosin-binding site. Methods: We produced four wild-type (WT) nebulin super repeats (S9, S14, S18, and S22), 283 to 347 amino acids long, and five corresponding repeats with a patient mutation included: three missense mutations (p.Glu2431Lys, p.Ser6366Ile, and p.Thr7382Pro) and two in-frame deletions (p.Arg2478_Asp2512del and p.Val3924_Asn3929del). We performed F-actin and tropomyosin-binding experiments for the nebulin super repeats, using co-sedimentation and GST (glutathione-S-transferase) pull-down assays. We also used the GST pull-down assay to test the affinity of WT nebulin super repeats for WT alpha- and beta-tropomyosin, and for beta-tropomyosin with six patient mutations: p.Lys7del, p. Glu41Lys, p.Lys49del, p.Glu117Lys, p.Glu139del and p.Gln147Pro. Results: WT nebulin was shown to interact with actin and tropomyosin. Both the nebulin super repeats containing the p.Glu2431Lys mutation and nebulin super repeats lacking exon 55 (p.Arg2478_Asp2512del) showed weak affinity for F-actin compared with WT fragments. Super repeats containing the p.Ser6366Ile mutation showed strong affinity for actin. When tested for tropomyosin affinity, super repeats containing the p.Glu2431Lys mutation showed stronger binding than WT proteins to tropomyosin, and the super repeat containing the p.Thr7382Pro mutation showed weaker binding than WT proteins to tropomyosin. Super repeats containing the deletion p. Val3924_Asn3929del showed similar affinity for actin and tropomyosin as that seen with WT super repeats. Of the tropomyosin mutations, only p.Glu41Lys showed weaker affinity for nebulin (super repeat 18). Conclusions: We demonstrate for the first time the existence of direct tropomyosin-nebulin interactions in vitro, and show that nebulin interactions with actin and tropomyosin are altered by disease-causing mutations in nebulin and tropomyosin.
  • Viita, Tiina; Kyheroinen, Salla; Prajapati, Bina; Virtanen, Jori; Frilander, Mikko J.; Varjosalo, Markku; Vartiainen, Maria K. (2019)
    In addition to its essential functions within the cytoskeleton, actin also localizes to the cell nucleus, where it is linked to many important nuclear processes from gene expression to maintenance of genomic integrity. However, the molecular mechanisms by which actin operates in the nucleus remain poorly understood. Here, we have used two complementary mass spectrometry (MS) techniques, AP-MS and BioID, to identify binding partners for nuclear actin. Common high-confidence interactions highlight the role of actin in chromatin-remodeling complexes and identify the histone-modifying complex human Ada-Two-A-containing (hATAC) as a novel actin-containing nuclear complex. Actin binds directly to the hATAC subunit KAT14, and modulates its histone acetyl transferase activity in vitro and in cells. Transient interactions detected through BioID link actin to several steps of transcription as well as to RNA processing. Alterations in nuclear actin levels disturb alternative splicing in minigene assays, likely by affecting the transcription elongation rate. This interactome analysis thus identifies both novel direct binding partners and functional roles for nuclear actin, as well as forms a platform for further mechanistic studies on how actin operates during essential nuclear processes. This article has an associated First Person interview with the first author of the paper.
  • Sidorenko, Ekaterina; Vartiainen, Maria K. (2019)
    Myocardin-related transcription factor A (MRTF-A) and serum response factor (SRF) form an essential transcriptional complex that regulates the expression of many cytoskeletal genes in response to dynamic changes in the actin cytoskeleton. The nucleoskeleton, a "dynamic network of networks," consists of numerous proteins that contribute to nuclear shape and to its various functions, including gene expression. In this review, we will discuss recent work that has identified many nucleoskeletal proteins, such as nuclear lamina and lamina-associated proteins, nuclear actin, and the linker of the cytoskeleton and nucleoskeleton complex as important regulators of MRTF-A/SRF transcriptional activity, especially in the context of mechanical control of transcription. Impact statement Regulation of gene expression is a fundamental cellular process that ensures the appropriate response of a cell to its surroundings. Alongside biochemical signals, mechanical cues, such as substrate rigidity, have been recognized as key regulators of gene expression. Nucleoskeletal components play an important role in mechanoresponsive transcription, particularly in controlling the activity of MRTF-A/SRF transcription factors. This ensures that the cell can balance the internal and external mechanical forces by fine-tuning the expression of cytoskeletal genes.
  • Santio, Niina M.; Vainio, Veera; Hoikkala, Tuuli; Mung, Kwan Long; Lång, Mirka; Vahakoski, Riitta; Zdrojewska, Justyna; Coffey, Eleanor T.; Kremneva, Elena; Rainio, Eeva-Marja; Koskinen, Päivi J. (2020)
    Background: The PIM family kinases promote cancer cell survival and motility as well as metastatic growth in various types of cancer. We have previously identified several PIM substrates, which support cancer cell migration and invasiveness. However, none of them are known to regulate cellular movements by directly interacting with the actin cytoskeleton. Here we have studied the phosphorylation-dependent effects of PIM1 on actin capping proteins, which bind as heterodimers to the fast-growing actin filament ends and stabilize them. Methods: Based on a phosphoproteomics screen for novel PIM substrates, we have used kinase assays and fluorescence-based imaging techniques to validate actin capping proteins as PIM1 substrates and interaction partners. We have analysed the functional consequences of capping protein phosphorylation on cell migration and adhesion by using wound healing and real-time impedance-based assays. We have also investigated phosphorylation-dependent effects on actin polymerization by analysing the protective role of capping protein phosphomutants in actin disassembly assays. Results: We have identified capping proteins CAPZA1 and CAPZB2 as PIM1 substrates, and shown that phosphorylation of either of them leads to increased adhesion and migration of human prostate cancer cells. Phosphorylation also reduces the ability of the capping proteins to protect polymerized actin from disassembly. Conclusions: Our data suggest that PIM kinases are able to induce changes in actin dynamics to support cell adhesion and movement. Thus, we have identified a novel mechanism through which PIM kinases enhance motility and metastatic behaviour of cancer cells.
  • Santio, Niina M; Vainio, Veera; Hoikkala, Tuuli; Mung, Kwan L; Lång, Mirka; Vahakoski, Riitta; Zdrojewska, Justyna; Coffey, Eleanor T; Kremneva, Elena; Rainio, Eeva-Marja; Koskinen, Päivi J (BioMed Central, 2020)
    Abstract Background The PIM family kinases promote cancer cell survival and motility as well as metastatic growth in various types of cancer. We have previously identified several PIM substrates, which support cancer cell migration and invasiveness. However, none of them are known to regulate cellular movements by directly interacting with the actin cytoskeleton. Here we have studied the phosphorylation-dependent effects of PIM1 on actin capping proteins, which bind as heterodimers to the fast-growing actin filament ends and stabilize them. Methods Based on a phosphoproteomics screen for novel PIM substrates, we have used kinase assays and fluorescence-based imaging techniques to validate actin capping proteins as PIM1 substrates and interaction partners. We have analysed the functional consequences of capping protein phosphorylation on cell migration and adhesion by using wound healing and real-time impedance-based assays. We have also investigated phosphorylation-dependent effects on actin polymerization by analysing the protective role of capping protein phosphomutants in actin disassembly assays. Results We have identified capping proteins CAPZA1 and CAPZB2 as PIM1 substrates, and shown that phosphorylation of either of them leads to increased adhesion and migration of human prostate cancer cells. Phosphorylation also reduces the ability of the capping proteins to protect polymerized actin from disassembly. Conclusions Our data suggest that PIM kinases are able to induce changes in actin dynamics to support cell adhesion and movement. Thus, we have identified a novel mechanism through which PIM kinases enhance motility and metastatic behaviour of cancer cells. Video abstract Graphical abstract
  • Zeinoddin, Narjes (Helsingin yliopisto, 2020)
    Endocytosis is the process responsible for internalising membrane components and as such plays a key role in the biology of this structure. Mammalian cells have evolved various endocytic strategies, but Clathrin-Mediated Endocytosis (CME) is the most common type. Since the discovery of CME, around 50 years ago, the field has built a remarkable wealth of knowledge on the core CME components. In stark contrast, our understanding on the relationship between CME and the actin cytoskeleton, which is present throughout the process, is still in its infancy. In this thesis, I show the production and characterisation of recombinant, SpyCatcher tagged transferrin (TF), a canonical CME ligand. TF was expressed in E. coli and using an optimised protocol, successfully solubilised and refolded from inclusion bodies. The protein was then labelled with a fluorophore and purified to a high level of purity. Tests in mammalian cells showed that home-made TF has the same endocytic behaviour as TF purified from human plasma. Moreover, I could show that the SpyCatcher moiety attached to our home-made TF is capable to mediate its covalent linkage to its counterpart SpyTag. The successful production, refolding and functional characterization of recombinant TF in this study is an important first step to examine the participation of the actin cytoskeleton during CME.
  • Hyrskyluoto, Alise; Vartiainen, Maria K. (2020)
    Actin has essential functions both in the cytoplasm and in the nucleus, where it has been linked to key nuclear processes, from transcription to DNA damage response. The multifunctional nature of actin suggests that the cell must contain mechanisms to accurately control the cellular actin balance. Indeed, recent results have demonstrated that nuclear actin levels fluctuate to regulate the transcriptional activity of the cell and that controlled nuclear actin polymerization is required for transcription activation, cell cycle progression, and DNA repair. Intriguingly, aberrant nuclear actin regulation has been observed, for example, in cancer, signifying the importance of this process for cellular homeostasis. This review discussed the latest research on how nuclear actin is regulated, and how this influences actin-dependent nuclear processes.
  • Jiu, Yaming; Peränen, Johan; Schaible, Niccole; Cheng, Fang; Eriksson, John E.; Krishnan, Ramaswamy; Lappalainen, Pekka (2017)
    The actin and intermediate filament cytoskeletons contribute to numerous cellular processes, including morphogenesis, cytokinesis and migration. These two cytoskeletal systems associate with each other, but the underlying mechanisms of this interaction are incompletely understood. Here, we show that inactivation of vimentin leads to increased actin stress fiber assembly and contractility, and consequent elevation of myosin light chain phosphorylation and stabilization of tropomyosin-4.2 (see Geeves et al., 2015). The vimentin-knockout phenotypes can be rescued by re-expression of wild-type vimentin, but not by the non-filamentous ` unit length form' vimentin, demonstrating that intact vimentin intermediate filaments are required to facilitate the effects on the actin cytoskeleton. Finally, we provide evidence that the effects of vimentin on stress fibers are mediated by activation of RhoA through its guanine nucleotide exchange factor GEF-H1 (also known as ARHGEF2). Vimentin depletion induces phosphorylation of the microtubule-associated GEF-H1 on Ser886, and thereby promotes RhoA activity and actin stress fiber assembly. Taken together, these data reveal a new mechanism by which intermediate filaments regulate contractile actomyosin bundles, and may explain why elevated vimentin expression levels correlate with increased migration and invasion of cancer cells.