During cell migration, the distribution of certain plasma membrane proteins, such as extracellular matrix (ECM) receptors (integrins) is highly polarised.
Extracellular matrix is a complex structural entity surrounding and supporting cells. The ECM is composed of 3 major classes of biomolecules:
1. Structural proteins: collagen and elastin.
2. Specialized proteins: fibrillin, fibronectin and laminin.
3. Proteoglycans: these are composed of a protein core to which is attached long chains of repeating disaccharide units termed of glycosaminoglycans (GAGs) forming extremely complex high molecular weight components of the ECM.
The cell before migration must adhere to ECM by cell adhesion molecules such as selectins, integrins, and cadherins. Correct cellular adhesion is essential in maintaining multicellular structure. Cellular adhesion can link the cytoplasm of cells and can be involved in signal transduction.
Cadherins (named for "calcium-dependent adhesion") are a class of type-1 transmembrane proteins. They play important roles in cell adhesion, ensuring that cells within tissues are bound together. They are dependent on calcium (Ca2+) ions to function, hence their name.
The cadherin superfamily includes cadherins, protocadherins, desmogleins, and desmocollins, and more.In structure, they share cadherin repeats, which are the extracellular Ca2+-binding domains.
There are many members of the classic cadherin family but E-cadherin in epithelial tissues has been the most studies in stable adhesions.
Continued expression and functional activity of E-cadherin are required for cells to remain associated in the epithelium.
E-cadherin is thought to act as an important suppressor of epithelial tumor cell invasiveness and metastasis. A loss of E-cadherin expression or function leads to enhanced cell invasiveness in cell culture,and E-cadherin deficiencies or mutations correlate with the invasiveness and metastasis of certain human tumors.(Cell adhesion: the molecular basis of tissue architecture and morphogenesis,1996).
Loss of E-cadherin function occurs during malignant progression in almost all epithelial cancers,
serving as a clinical indicator for poor prognosis and metastasis. In many cases, its functional loss is caused by germline and somatic gene mutations, chromosomal aberrations,transcriptional repression, and DNA hypermethylation of the E-cadherin (cdh1) gene.
A large number of growth factors and their activated signal transduction pathways are known to provoke the loss of E-cadherin function and to induce cancer cell migration and invasion,including transforming growth factor β (TGFβ), hepatocyte growth factor (HGF), members of the epidermal growth factor (EGF) family, insulin-like growth factor (IGF), fibroblast growth factor (FGF), and Notch signaling.
In most epithelial cancers, the loss of E-cadherin function during tumor progression results in an increased expression of the mesenchymal cadherin, N-cadherin (and sometimes other mesenchymal cadherins), with a drastic change in the adhesive properties of cancer cells, as they lose their affinity for epithelial neighbors and gain affinity for stromal cells. The cadherin switch by itself seems to provoke cell migration and invasion.(Mechanisms of motility in metastasizing cells,2010).
To regulate motility of cell by rearrangement of cytoskeleton there is the Rho-family of p21 small GTPases that can interact with downstream effector molecules to propagate the signal transduction in their GTPloaded “on” state. The intrinsic phosphatase activity hydrolyzes the GTP to GDP, turning the protein “off”. This process is accelerated by the interaction with GAPs (GTPase activating proteins). The interaction with GEFs (guanine-nucleotide exchange factors) facilitates the exchange of GDP to GTP.
Traditionally, the Rho family GTPases, RhoA, Rac1 and Cdc42 have been primarily associated with cytoskeleton rearrangements. RhoA causes stress fiber and adhesion formation, Rac1 induces sheet-like lamellipodial protrusion and Cdc42 produces filopodial protrusions. Dynamics of the Rho-family small GTPases in actin regulation and motility,2010 .
Evidences indicate that RhoA contributes to the malignant properties of cancer cells. This is counterbalanced by the negative effects that hyperactivated RhoA may have in cell migration. In fact RhoA hyperactivated induces fibers to stress and induces focal adhesions that inhibit polarity and migration or, alternatively, the stimulation of abnormally high levels of actomyosin contractility that could lead to cell detachment and anoikis.
To deal with this problem, cancer cells have implemented a number of negative regulatory steps to tune down the activity of the RhoA pathway. Those include, among others, the release of RhoA from the plasma membrane via phosphorylation by protein kinase A (PKA) and protein kinase G (PKG), the proteosomal degradation of RhoA induced by E3 ubiquitin ligases (Smurf1, Cullin), the interference with the RhoA activation cycle via inhibition of RhoA guanosine nucleotide exchange factors (GEFs) and stimulation of GTPase activating proteins (GAPs), the obstruction of effector binding by the previous interaction of RhoA with cell cycle inhibitors (p27Kip1), or the inhibition of Rock activity by the direct interaction of this serine/threonine kinase with GTPases (RhoE/Rnd3,Gem, R-Rad) and cell cycle inhibitors.
A transcriptional cross-talk between RhoA and c-Myc inhibits the RhoA/Rock-dependent cytoskeleton,2010
Schematic representation of RhoA signaling elements connected to the regulation of the F-actin cytoskeleton and known inhibitory steps operating in this route. Signaling proteins are shown in green. Transcriptional factors are depicted in yellow. Molecules that inhibit either RhoA or Rock are shown in blue. Activation and inactivation steps are shown as arrows and blunted lanes, respectively. Stress fibers, focal adhesions and nuclei are depicted in red, green and yellow, respectively.
Rho-associated protein kinase (ROCK) is a kinase belonging to the AGC (PKA/ PKG/PKC) family of serine-threonine kinases. It is mainly involved in regulating the shape and movement of cells by acting on the cytoskeleton.
ROCK plays a role in a wide range of different cellular phenomena, as ROCK is a downstream effector protein of the small GTPase Rho, which is one of the major regulators of the cytoskeleton.
1. ROCK is a key regulator of actin organization and thus a regulator of cell migration as follows:
Different substrates can be phosphorylated by ROCKs, including LIM kinase, myosin light chain (MLC) and MLC phosphatase. These substrates, once phosphorylated, regulate actin filament organisation and contractility as follows:
ROCK inhibits the depolymerisation of actin filaments indirectly, ROCK phosphorylates and activates LIM kinase, which in turn phosphorylates ADF/cofilin, thereby inactivating its actin-depolymerization activity. This results in the stabilization of actin filaments and an increase in their numbers. Thus, over time actin monomers that are needed to continue actin polymerization for migration become limited. The increased stable actin filaments and the loss of actin monomers contribute to a reduction of cell migration.
ROCK also regulates cell migration by promoting cellular contraction and thus cell-substratum contacts. ROCK increases the activity of the motor protein myosin II by two different mechanisms:
firstly, phosphorylation of the myosin light chain (MLC) increases the myosin II ATPase activity. Thus several bundled and active myosins, which are asynchronously active on several actin filaments, move actin filaments against each other resulting in the net shortenting of actin fibres.
Secondly, ROCK inactivates MLC phosphatase, leading to increased levels of phosphorylated MLC.
Thus in both cases, ROCK activation by Rho induces the formation of actin stress fibres, actin filament bundles of opposing polarity, containing myosin II, tropomyosin, caldesmon and MLC-Kinase, and consequently of focal contacts, which are immature integrin-based adhesion points with the extracellular substrate.
Rho-associated protein kinase
Migration requires polarized plasma membrane proteins according to the direction of travel.
Plasma membrane components may be maintained in a polarised state either by a diffusion barrier or by an active endo-exocytic process that retargets protein to particular regions of the cell surface.
Distruption of endosomal transport and/or of the exocytic fusion of recycling vescicles can compromise polarity during cell migration (Depletion of intracellular potassium disrupts coated pits and reversibly inhibits cell polarization during fibroblast spreading, 1993).
The integrin is distributed more on the lower surface of the cell and how vescicular transport acts to drive integrin toward the advancing lamellipodium is not completely clear.
The integrin family of cell adhesion receptors comprises 24 distinct alphabeta heterodimers that recognize glycoprotein ligands in the extracellular matrix or on cell surfaces. Integrins and their ligands play fundamental roles in all events that involve cell adhesion, detachment and migration, the hallmarks of multicellular organisms. Strict and dynamic control of the integrin's affinity for ligand by cellular mechanisms is of crucial importance, since ligand binding results in integrin signaling with the potential of changing the cell's fate. Aberrant interactions of integrins with their ligands have been implicated in many pathophysiological states. Therefore elucidation of the mechanisms for ligand recognition and signaling induced by ligand binding is key to the development of effective drugs against numerous disease processes including cancer metastasis, thrombosis and autoimmunity.
Many members of the integrin family, including alpha5beta1, alpha8beta1, alphaIIbbeta3, alphaVbeta3, alphaVbeta5, alphaVbeta6 and alphaVbeta8, recognize an Arg–Gly–Asp (RGD) motif within their ligands. These ligands include fibronectin (Fn), fibrinogen, vitronectin.(Structure of integrin alpha5bold beta1 in complex with fibronectin,2003).
Integrin may be endocytosed at the rear of the cell following focal adhesion disassembly and then transported forwards within vescicles for re-exocytosis at the cell front but there isn't no evidence that endocytic rates are augmented at the cell rear.
In fact most studies indicate increased rates of internalisation and localisation of clathrin-dependent endocytosis toward the leading edge of migrating cells (Real-time analysis of clathrin-mediated endocytosis during cell migration,2003).
Endocytosis is regulated by Dynamin by both clathrin-dependent and independent mechanisms and mediates microtubule-driven focal complex disassembly.(Microtubule-induced focal adhesion disassembly is mediated by dynamin and focal adhesion kinase, 2005).
Integrin-containing vescicles are seen to move backwards from the leading edge toward the recycling endosomes located in the perinuclear region indicating that internalised material initially follows a retrograde path.
Endocytic recycling pathways: emerging regulators of cell migration, 2006.
In the picture b we can see dynamin-driven endocytosis at leadinge edge and retrograde transport of endosomes.
MTOC is microtubule organising centre;
RE are recycling endosomes;
After internalisation to early endosomes (EEs), receptors are transported to multivescicular bodies and then to late endosomes for degradation or they are recycled to the plasma membrane.
Some receptors such as alphav-beta3 integrin, return to the plasma membrane from early endosomes under the control of the Rab 4 GTPase and this is the "short loop".
Receptors not returned via the short-loop pathway traffic from EEs (early endosome) to the perinuclear recycling compartment (PNRC) recycle via a "long-loop" pathway that can be regulated by Rab 11.
Arfs1 and 6 may collaborate with Rab 11 to control recycling from the PNRC.
Rab and Arf GTPases may exert their effects on membrane transport via interactions with specific effector proteins.
Endocytic recycling pathways: emerging regulators of cell migration, 2006.
Rab 11 is upregulated during skin carcinogenesis (c-Fos-Dependent Induction of the Small Ras-Related GTPase Rab11a in Skin Carcinogenesis, 2005 ), is linked to Barrett's dysplasia (Rab11 in dysplasia of Barrett's epithelia, 1999) and it is involved in hypoxia-promoted invasive transmigration of carcinoma cells(Hypoxia stimulates carcinoma invasion by stabilizing microtubules and promoting the Rab11 trafficking of the alpha6beta4 integrin, 2005 ).
These tumorigenic effects of Rab 11 are due to its ability to directly control integrin trafficking.
The recycling of Alphav-beta3, Alpha5-beta1 and Alpha6-beta4 integrins require PKB/Akt.
in fact PKB/Akt influences trafficking of Beta1 integrin via the PNRC by phosphorilation of ACAP1 (Phosphorylation of ACAP1 by Akt regulates the stimulation-dependent recycling of integrin beta1 to control cell migration, 2005 ).
ACAP1 is a GAP for Arf6 and when it is phosphorilated by PKB/Akt, promotes recycling of Beta1 integrin by associating with the Beta1 cytotail at the recycling endosome.
Integrin heterodimers are crucial mediator of many cell-extracellular matrix and cell-cell interactions.
Many studies had proposed that the observed internalization and recycling of integrins implied that endocytosis is required in cell migration.
Between the kind of integrins which are internalised by clathrin-dependent mechanisms, there is a sub population of Beta1 integrins can follow internalization routes that are regulated by the AP-2 clathrin adaptor.
Endocytic transport of integrins during cell migration and invasion,2008
Mutations of the AP-2 adaptor-binding motif prevents internalization.
This mutation inhibites only a fraction of Beta1, only Beta1 which are associated with CD151.
The association of polarized clathrin-mediated endocytosis of integrins to cell migration has been provided by studies about Numb.
Numb is a cargo-specific endocytic adaptor that interacts with components of the clathrin endocytic machinery.
Numb can bind directly to Beta-integrin subunits by its phosphotyrosin domain (PTB) and this co-localises with Beta integrin in clathrin-coated structures (CCs) surrounding adhesion sites at the leading edge of HeLa cells (Numb controls integrin endocytosis for directional cell migration with aPKC and PAR-3, 2007 ).
Polarised endocytosis of integrin is imparted through the ability of Numb to recruit the polarity protein PAR-3, which recruits aPKC and the phosphorilation of Numb by PKC renders it enable to associated with integrins.
Recycling of Beta1 integrins from the PNRC is known to be controlled by Rab11 in a way that requires the activity of pro-migratory kinases such as PKC and PKB/Akt (PKC epsilon controls the traffic of beta1 integrins in motile cells, 2002 ) which phosphorilates the cytoskeletal protein vimentin and the ARF6 GTPase activating protein GAP ACAP1.
More recently, research has demonstrated that ACAP1 mediates the connection between PKB and integrin recycling (Phosphorylation of ACAP1 by Akt regulates the stimulation-dependent recycling of integrin beta1 to control cell migration, 2005).
ACAP1 when is phosphorilated by PKB binds directly to Beta1 integrin on endosomal membranes and this promotes its recycling.
The phosphorilation of ACAP1 by PKB is required for cells to migrate and this finding suggests that ACAP1-dependent stimulated recycling of Beta1 integrin plays a role in cell migration.
ACAP1 interacts directly with clathrin heavy chain (An ACAP1-containing clathrin coat complex for endocytic recycling, 2007 ) and this interaction doesn't seem to regulate integrin internalization.
ARF6 regulates the trafficking of internalized membrane and receptors towards the plasma membrane and also ARF6 has been found at the plasma membrane where it has a role in the reorganization of the cytoskeleton and in endocytosis (ARF proteins: roles in membrane traffic and beyond, 2006 ).
Inhibition of ARF6 is reported to suppress Beta1 integrin recycling and to lead to an accumulation of Beta1 integrins within intracellular endosomes.
Pools of ARF6 at intracellular membranes and at the plasma membrane are controlled by different GEFs, in particular GEF-100 and BRAG2 that not only controls Beta1 integrin trafficking but also interacts with EGFR and transduces signals that activate ARF6 and promote invasion and metastasis.
Endocytic trafficking of integrins contributes to invasive migration.
Rab11 is involved in tumorogenesis in particular in hypoxia-induced invasion which also requires Alpha6-beta4 integrin.
Overexpression of the Rab11 family member Rab25 is strongly associated with aggressive cancers (The RAB25 small GTPase determines aggressiveness of ovarian and breast cancers, 2004 ).
Rab25 binds to the Beta1 subunit of Alpha5-beta1 and in this way regulates it (Rab25 associates with alpha5beta1 integrin to promote invasive migration in 3D microenvironments, 2007 ).
This receptor–ligand pair is functionally very important because it mediates fibronectin fibril formation and governs extracellular matrix assembly, which is vital to cell function in vivo.(Structure of integrin alpha5bold beta1 in complex with fibronectin, 2003).
Alpha5-beta1 integrin molecular structure:
Alpha5-beta1 integrin genes sequence databases:
In the absence of Rab25, tumor cells do not produce pseudopodia and Alpha5-beta1 is free to move from the front towards the rear of the cell.
When tumor cells expressing Rab25 invade a collagen hydrogel containing fibronectin, their movements is characterized by the extension of a long pseudopod in the direction of migration.
Alpha5-beta1 integrin in this case is localized to the plasma membrane and to Rab25-positive endosomes that reside near the tip of the extending pseudopod.
A pool of Alpha5-beta1 at the pseudopodial tip cycles rapidly between the Rab25 compartment and the plasma membrane, and that this prevents rearward movement of the integrin.
Endocytic transport of integrins during cell migration and invasion, 2008
Today we are beginning to be able to assemble a picture of how and why particular integrin endocytosis and recycling events contribute to cell migration in a range of cell types and phisiological contexts.
For the future a key to determine how integrins are shuttled between endosomes and the plasma membrane within restricted cellular locales (such as pseudopodial tip region) therein dictate signalling events controlling actin dynamics, in fact the actin polymerisation machinery is the most route to regulating processes that determine the protrusive activity of invasive structures as cancer cells migrate through 3D matrices.