M2L3 Cytoskeletal regulation

Overview of types of cytoskeleton

  • Microtubules

    • Long structures involved in organelle and vesicular transport, cell polarity, cell division, and structural support

    • Made of α and β subunits, forming a tubule stucture

  • Intermediate filaments

    • Found everywhere, varying in shape and size depending on location

    • Provides mechanical strength, resistance to sheer stress, cell shape maintenance, anchorage of organelles

    • Many types - neurofilaments, vimentin, keratin, lamins, desmin etc

  • Actin filaments

    • Involved in cell movement/migration, muscle contraction, membrane organisation, organisation of membrane receptors/proteins

    • Cell migration in tumour invasion and metastasis is facilitated by the constant turnover of the actin cytoskeleton 

Actin polymerisation

  • Whole cell motility and mechanosensing relies of continual restructuring of actin cytoskeleton

  • Actin filament depolymerisation ensures turnover of actin filaments and maintains a pool of monomers to recycle for polymerisation

  • Actin is made of monomeric globular actin, which are highly energy dependent

    • Globular actin binds to ATP to form ATP-actin

    • Three ATP-actin monomers come together to form the nucleation site, from which polymerisation occurs

      • Profilin promotes the assembly of actin filaments, by catalysing the exchange of ADP for ATP, and inhibits spontaneous nucleation

    • ATP hydrolysis converts ATP-actin to ADP-actin for depolymerisation 

      • Cofilin severs actin filaments so that they do not need to get released one by one, accelerating depolymerisation

    • Actin polymerisation coupled with depolymerisation is known as actin treadmilling, resulting in a highly dynamic cytoskeleton

  • Nucleation factors help to form the actin filament, which is normally energetically unfavourable

    • Arp2/3 complexes are the most well-studied - it is a controllable nulceating structure which binds together G-actin monomers to initiate polymersation or binds to existing actin subunits at the ‘mother’ filament to form ‘daughter’ filament branches

    • Formins also initiate linear subunit addition at the + end of actin and its FH2 domain stabilises a spontaneously formed actin dimer to nucleate a new filament, elongating it by recruiting more actin monomers using its FH1 domain and profilin 

    • There are more actin nucleators which have WH2 domains

  • Elongation factors (eg. VASP) help to formed actin filaments and stabilise the structure to aid in elongation

  • Nucleation and elongation factors were initially identified in patients with Wiskott-Aldrich syndrome (characterised by bleeding, eczema, expression in HSCs)

    • They are thus known as WAS proteins (WASp)

    • Good at allowing actin to polymerise, responding to extracellular signals, regulating cell migration, and are expressed as five different isoforms depending on where they are expressed (WASp, N-WASp, WAVE1, WAVE2, WAVE3)

  • Thymosins sequester actin monomers to which they bind and block filament assembly and release them when filament growth is needed

Cell migration

  • Membrane protrusion (extension of filopodia and lamellipodia)  → focal adhesion → translocation (loosening of focal adhesions at the rear) → retraction of trailing edge

  • Facilitated by Rho family GTPases (belonging to the Ras superfamily)

    • Rho-GTP is the active form while Rho-GDP is the inactive form (molecular switch)

    • Rho family includes Cdc42, Rho and Rac

  • At the leading edge of the cell, the high rate of actin polymerisation is faciliated by Cdc42 and Rac, leading to the formation of:

    • Filopodia - finger-like, made of parallel F-actin bundles

    • Lamellipodia - flat regions with highly branched actin network

    • Invadopodia - projection into the ECM for remodelling (eg. in carcinoma)

    • Podosomes - projections into ECM (but do not necessarily remodel) (eg. monocytic cells)

  • All of these structures generate locomotive force 

  • Translocation and retraction is largely mediated by Rho

    • Active Rho (Rho-GTP) activates Rho trigger (ROCK), which phosphorylates downstream targets to activate myosin and trigger actomyosin contractile forces for locomotion

  • In collective migration in cancer, sheets of cells can move together

    • Migrating adherent vs migrating leader-follower models

  • Cells can also migrate individually

    • The migrating adherent model and individual migration model involve a cadherin shift (EMT)

  • Modes of migration

    • Mesenchymal (cells take on an elongated. fibroblast-like morphology)

      • More common in dense matrices

      • Involving extension of invadopodia and proteolytic ECM remodeling using MMPs in an integrin-dependent manner

      • Creates a proteolytic path for migration and invasion

    • Amoeboid (cells have rounded morphology)

      • More common in less dense matrices, involving contraction of cortical actomyosin cytoskeleton to create hydrostatic pressure needed to squeeze the cell body through gaps in the matrix

      • Integrin-independent process

      • Eg. non-neoplastic cells (lymphocytes, neutrophils) or neoplasmic cells (eg. lymphoma, leukaemia, small cell lung carcinoma)

    • Balance between Rac and Rho determine the type of migration

      • Mesenchymal - Rac dependent

      • Amoeboid - Rho/ROCK signalling dependent, actin cytoskeleton reorganised at PM with dynamic blebbing

Role of RASSF1A in nuclear actin regulation and cell mechanotransduction

  • Tumours are highly stiff compared to surrounding tissue due to highly fibrous ECM

  • ECM can signal to cells to produce cytokines and chemokines to help maintain its stiffness

    • TGFβ and IL-15 are particularly good at signalling to the cells

    • TGFβ in particular is notable for inducing EMT and cytoskeletal reorganisation

  • To respond to mechanical signals, cells:

    • Activate mechanosensors - detect cues such as force, stress, strain etc.

      • Detected by integrins, GPCRs, TRP ion channels, mechanosensitive Piezo ion channels, YAP/TAZ regulators of mechanotransduction

    • Initiate mechanotransduction - triggers signalling pathways

      • PI3K/AKT - cell survival and metabolism 

      • JAK/STAT - transmits signals from mechanical stimuli to alter gene expression

      • FAK - mediates cell adhesion and signalling

      • Hippo - regulates growth and organ size

      • RhoA - activates responses to mechanical stress via GTP-binding proteins

    • Alter cell functions (altering gene expression, cytoskeleton organisation, membrane traffic)

  • Hippo signalling is largely involved in cell proliferation and development

    • Involves the kinase MST2 and SAV1 and RASSF1A apaptors which help activate MST2

    • When Hippo pathway is active MST2 phosphorylates LATS1/2 and MOB1A/B

    • LATS1/2 phosphorylates YAP/TAZ which get retained in the cytoplasm or targeted for degradation

    • When there is Hippo signalling dysregulation (common in cancer), LATS1/2 is not phosphorylated and YAP/TAZ is able to enter the nucleus

    • YAP/TAZ activates TEAD1-4 transcription factors which activate growth and survival genes

  • YAP/TAZ are well known mechanosensors

    • When they are active (stiff ECM, cell spreading, high contractile force), YAP/TAZ enter the nucleus and activate TEAD to promote cell proliferation in cancer or osteogenic   differentiation

    • When inactive (confined cell adhesion, soft ECM, low contractile force), YAP/TAZ are proteasomally degraded, causing apoptosis, growth arrest, or adipocyte differentiation

  • Triggering Rho/Rac pathways and F-actin production can prevent LATS1/2 phosphorylation, allowing YAP/TAZ-mediated activation of proliferation and survival genes by interacting in the Hippo pathway

  • RASSF1A is epigenetically silenced in almost all solid cancers

    • Loss of RASSF1A —> matrix stiffness, collagen deposition, metastasis,

      • Also higher levels of stem cell TF Nanog

      • Increased stiffness activates Wnt/β-catenin and stemness

      • MST2 recruits RASSF1A to the nuclear envelope and RASSF1A interacts with XPO6/RAN to export G-actin, so loss of RASSF1A leads to increase in G-actin in the nucleus

        • XPO6 involved in shuttling G-actin from nucleus to cytoplasm and IPO9 brings it back

        • Therefore RASSF1A regulates nuclear actin

  • In the nuclear envelope there are LINC complexes, made of SUN proteins, and KASH proteins (nesprins) which act as bridge to bind to different cytoskeletal elements depending on the type/location of the nesprin

    • Mechanical signals get transmitted to the nucleus via LINC complexes

    • When the signal is transduced, formins are recruited or coordinated to promote localised actin polymerisation in the nucleus

  • ATR is responsible for phosphorylating RASSF1A

  • RASSF1A can bind with Filamin A to regulate nuclear actin

  • Nuclear actin can regulate chromatin accessibility, EMT, DNA repair and CD4+ T cell effector functions

  • T cells and NK cells have very high RASSF1A expression and activated T cells have increased nuclear actin, responsible for Ca2+ signalling and effector responses (antibody production, antigen recognition)

    • RASSF1A loss impairs T cell migration and reduces T ccell tumour killing efficiency in CD8+ T cells due to downregulation of CD69 and nuclear actin

  • T cells have many microvilli to scan surfaces and they are rich in adhesion molecules and TCR, they are used to established contacts and initiate signalling

    • Steps in establishing contacts: searching, scanning, signalling, spreading, synapsing

    • Microvilli shape is disrupted in T cells with RASSF1A mutations which impairs its function, not making contacts with tumour cells