Cell Junction and Cytoskeleton-related signaling

Tight Junctions

As adhesion complexes in epithelial and endothelial cells, tight junctions act first and foremost as barriers, tightly regulating permeability by controlling movement of molecules in the space between cells and across tissues by size and charge (1). They also form an intramembrane diffusion barrier which segregates the part of the membrane attached to the extracellular matrix (basolateral membrane) from the part exposed to free fluid (apical plasma membrane).

Tight junctions exist as clusters of integral membrane proteins, which include Crumbs3 (CRB3), claudins, TJ-associated Marvel proteins (including Occludin, MarvelD3 and Tricellulin) and members of the immunoglobulin superfamily (IgSF), including the junction adhesion molecules (JAMs) (2). Each tight junction component can interact with intracellular scaffolding proteins that connect them to cytoskeletal components. The most prominent scaffolding proteins include the F-actin interacting zonula occludens (ZO) proteins ZO-1, ZO-2 and ZO-3 and the cingulin family proteins (including CGN and CGNL1/JACOP) that interact with microtubules in addition to F-actin. Different cluster compositions are associated with different tight junction functions, with claudins regulating permeability via charge and JAMs regulating permeability via size.

Beyond their classic function, tight junction proteins can also influence cellular processes including polarity, differentiation, growth and proliferation, plus cell motility and migration with aberrant activity being associated with multiple cancers.

Gap Junctions

In vertebrates, gap junctions are hexameric intercellular channels formed by a wide variety of proteins of the connexin family (3). These clusters or aggregates enable direct cell-cell communication by the transfer of ions and small molecules between cells, including second messengers like calcium, cAMP and inositol triphosphate (IP3). The connexin composition, which can be homomeric or heteromeric, results in different physical properties. Although gap junctions were first identified in voltage excitable cells such as neurons and muscle cells, they are present at the junctions of nearly all types of cells.

Mutations in connexin proteins have revealed a number of specialized functions and are associated with channel communication-opathies including deafness, hereditary cataracts and neuropathies including Charcot Marie Tooth (4).

Adherens junctions

Adherens junctions are primarily responsible for cell-cell adhesion and mechanical strength within tissues. They are composed of clusters of interacting cadherin transmembrane proteins on adjacent epithelial cells that form in response to nectin- and Afadin-induced cytoskeletal reorganization (5). Inside cells, cadherin clusters are linked to the actin cytoskeleton through adaptor proteins including α-catenin and β-catenin. Another important linker is vinculin, a mechanosensitive protein that is activated by tension to strengthen adhesion (6).

Integrin signaling

Integrins are a large family of heterodimeric transmembrane receptors that are crucial for mediating cell-extracellular matrix (ECM) and cell-cell interactions. Composed of membrane spanning alpha and beta subunits that mix and match to regulate different processes, integrins are activated by both “inside-out” and “outside-in” signals (7).

β-integrins generally have a short cytoplasmic tail that is accessible within the cytoplasm. Intracellular signals promote clustering and binding of intracellular adaptor proteins like Talin1 and Kindlin to the β-integrin tail. Binding triggers a conformational switch that enables the adaptors to interact with F-actin and is essential for integrin-dependent attachment and formation of focal adhesions – adhesion complexes that provide an anchor to the ECM. At the same time, conformational changes within the receptor itself result in “inside-out” increased affinity for ECM ligands.

Engagement of integrin receptors with extracellular ligands triggers “outside-in” signaling and activates formation of complexes of scaffold/adaptor proteins including paxillin, FAK and ILK that promote assembly of a variety of signaling components. Signaling through these scaffold/adaptor complexes at focal adhesions regulates several key cellular processes including growth factor-induced mitogenic signals, cell survival, cell proliferation and migration, cell locomotion and regulation of cell cycle.

Paxillin is an important adaptor protein that acts as a hub that connects structural and signaling components of the cell, coordinating downstream signaling through multiple other proteins (8). While it can be difficult to tease apart the exact recruitment steps, FAK, also known as focal adhesion kinase or PTK2, is one of the main partners of paxillin signaling. FAK recruits and forms a complex with Src tyrosine kinase which acts through JNK, MAPK and PI3K/Akt pathways among others to influence cytoskeletal organization, cell proliferation and more.

Paxillin additionally influences tension through its interaction with GIT1 (G protein-coupled receptor kinase-interacting protein 1) which complexes with guanine nucleotide exchange factors to target CDC42 and Rac/Rho.

Intricately linked with paxillin and FAK is a third scaffold/adaptor protein, ILK (integrin-linked kinase), that also plays a role in downstream PI3K/Akt, CDC42 and Rac/Rho signaling (9). Originally identified as a serine-threonine kinase, it is now believed that ILK may be a pseudo-kinase, which like paxillin, acts only as a scaffold protein.

FAK is upregulated in a wide variety of human epithelial cancers and is implicated in the progression of tumor cells to malignancy and the pathogenesis of cancer, suggesting opportunities for the development of anti-cancer therapeutics (8). Paxillin and ILK overexpression are similarly linked to properties of tumorigenesis and ultimately multiple cancers.

References

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2. Wibbe N, Ebnet K. Cell adhesion at the tight junctions: New aspects and new functions. Cells. 2023;12(23):2701.
3. Beyer EC, Berthoud VM. Gap junction gene and protein families: Connexins, innexins, and pannexins. Biochim Biophys Acta Biomembr. 2018;1860(1):5–8
4. Evans WH, Martin PEM. Gap junctions: structure and function (Review). Mol Membr Biol. 2002;19(2):121–36.
5. Garcia MA, Nelson WJ, Chavez N. Cell-cell junctions organize structural and signaling networks. Cold Spring Harb Perspect Biol. 2018;10(4):a029181.
6. Mui KL, Chen CS, Assoian RK. The mechanical regulation of integrin-cadherin crosstalk organizes cells, signaling and forces. J Cell Sci. 2016;129(6)1093–1100.
7. Li S, Sampson C, Liu C, Piao H, Liu H. Integrin signaling in cancer: Bidirectional mechanisms and therapeutic opportunities. Cell Commun Signal. 2023;21(1):266.
8. Ripamonti M, Wehrle-Haller B, de Curtis I. Paxillin: A hub for mechano-transduction from the β3 integrin-talin-kindlin axis. Front Cell Dev Biol. 2022;10:852016.
9. Górska A, Mazur AJ. Integrin-linked kinase (ILK): The known vs. the unknown and perspectives. Cell Mol Life Sci. 2022;79(2):100.