Introduction
Biological organisms are incredibly complex systems, capable of completing a wide array of actions and behaviors, mostly through the creation of specialized tissues which work in synchrony to produce these complex actions. A simple example would be locomotion, which requires multiple tissue types such as bone, muscle, and connective tissue. Each tissue’s properties are integral to its function, and without this variety in tissue type and properties, many complex biological behaviors could not exist. These properties arise not only from the cells within the tissues, but from the matrix of proteins on which they grow, known as the extracellular matrix (ECM). This matrix is a collection of proteins and small molecules, whose properties can vary greatly based on its exact composition. (Manou et al., 2019). Cells communicate back and forth with the ECM through protein complexes known as focal adhesions (FAs) (Wehrle-Haller et al., 2012). In this review I will provide a basic introduction to the ECM and FAs to highlight how cell-ECM interactions are a critical component of proper tissue differentiation and function, with a particular focus on how these interactions are relevant in a neuroscience setting. Rather than provide a fully comprehensive breakdown on the topic, this review should serve to provide a framework for understanding how these factors may affect cells, and expand the neuron-centric neuroscience curricula present at many institutions.
Basics of the Extracellular Matrix
In vivo, cells are not just packed in an array, but rather reside on a complex matrix of proteins which provides both the necessary “stuff” for cells to live on, and acts as a transmitter of information for the cells within this extracellular matrix (ECM) (Manou et al., 2019). This matrix is composed partially of proteins synthesized by the cell, which are interconnected with each other, but also connected to the cells within the matrix. This physical network allows for bidirectional regulation and signaling between cells and the ECM which enables biochemical information to move throughout this network (Bonnans et al., 2015). This matrix also dictates much of the chemical and physical properties of the tissue it composes, which is determined mostly by the concentrations of each ECM component. Here I will discuss the major components which could be considered the foundation or skeleton of the ECM and how their concentration affects the properties of the matrix as a whole.
The most abundant protein in most vertebrate ECMs is the collagen superfamily, containing 28 different types, which serve to create a rigid filamentous network (Sorushanova et al., 2019). Collagens are generally identified by their triple helical structure, which imparts a resistance to stretching to the collagen strand, making it a skeleton upon which the rest of the ECM can be built (Shoulders and Raines, 2009). The concentration of collagen is proportional to the rigidity of the medium, with higher concentrations being more rigid (Kadler et al., 2007). Due to its prevalence, there are many examples of collagen in vivo, but a possibly unappreciated role is in thrombosis and hemostasis, where collagen serves as an insoluble scaffold for other vascular proteins and cells (Farndale et al., 2004).
Proteoglycans are another large component of the ECM, and are composed of a core protein which holds multiple glycosaminoglycans (GAGs), with the exact quantity and composition varying depending on the specific proteoglycan, which impart a large negative charge to the molecule (Theocharis and Karamanos, 2019). This charge causes counterions and associated water molecules to be “absorbed” by the proteoglycans, imparting a more gel-like texture, which provides some resistance to compression (Zhao et al., 2013). The large variation of possible GAGs that compose a proteoglycan makes it useful in regulating tissue development. This is notable in the brain, where highly regulated spatio-temporal expression of proteoglycans plays a critical role in proper brain development, migration, axonal pathfinding, synaptogenesis, and plasticity (Schwartz and Domowicz, 2018).
A finer, mesh-like structure is also present in the form of laminins, a cross-shaped trimeric protein with binding sites for collagen and other compounds (Durbeej, 2010). Due to its relatively smaller size, laminin is the predominant anchor for cells to the ECM and as such plays a critical role in ECM morphogenesis (Miner and Yurchenco, 2004). This has been observed in the nervous system, with microglial morphology being affected by laminin in vitro (Tam et al., 2016).
These proteins interact with each other and cells through the help of fibronectins, a covalently bonded symmetrical dimer with a large array of binding sites which can link the ECM to a cell or other ECM components (Pankov and Yamada, 2002). Fibronectin can often move along fibrillar structures to help cell migration and matrix rearrangement (Dallas et al., 2006). Knocking out fibronectin in mouse embryos was lethal before embryogenesis, but homozygous fibronectin mutants showed widespread defects in mesoderm, neural tube, and vascular development. The observed deformities in neural tube and vascular structure seem to reinforce fibronectin’s key role in cell adhesion and migration (George et al., 1993)
A common feature of the ECM proteins mentioned, besides proteoglycans, is the RGD loop, named after its sequence (Arg-Gly-Asp), and functions as a critical binding site for cell adhesion (Ruoslahti, 1996).
Varying concentrations of ECM compounds will alter ECM properties, such as rigidity. These changes in rigidity can be critical in cell proliferation, morphogenesis, and differentiation (Tilghman et al., 2010). While historically the brain was thought to lack an ECM, persistent research identified not only its presence, but its significance in proper brain development and maintenance as well. Neuronal ECM contains a much lower concentration of collagen relative to other ECMs, and composes 15-20% of the brain’s volume (Lei et al., 2017). Rather than collagen, the neuronal ECM is composed primarily of proteoglycans, which are thought to help synaptic plasticity (Lau et al., 2013). However, the aforementioned role of collagen in maintaining vascular integrity is conserved in the blood-brain barrier (Thomsen et al., 2017). While neurons have been shown to be able to bind directly to laminin even in the absence of fibronectin (Liesi et al., 1984), fibronectin supports neurite outgrowth and axonal regeneration (Tonge et al., 2012). Although research on neuronal ECM has steadily increased, there is still much to be uncovered.
Basics of Cell-Matrix Interactions
Focal adhesions (FAs) are multi protein complexes that serve as a transductor of information, both mechanical and chemical, across the membrane, while also functioning as an anchor or linker for the cell to the ECM (Wehrle-Haller et al., 2012). Since there is such a wide range of proteins involved in FAs, I will only be discussing some of the more critical and well-studied proteins.
Integrins are a class of transmembrane heterodimeric proteins composed of 24 unique combinations from 18 ⍺-subunits and 8 𝛽-subunits and serve as the primary transmitters of information across the membrane (Kechagia et al., 2019). Integrins adopt an inactive conformation when unstimulated in which the cytoplasmic tails are bound. Talin binding is observed to be a final common step in integrin activation, since it is critical for triggering structural change in the protein. (Tadokoro et al., 2003). Additionally, the kindlin protein also serves to facilitate integrin activation by binding to the 𝛽-subunit cytoplasmic tail. (Rognoni et al., 2016). When activated integrins can identify a wide variety of ECM compounds, which is primarily determined by the specific combination of ⍺- and 𝛽-subunits (Hynes, 2004). A common binding site integrin is the RGD loop which is notably found in both laminin and fibronectin within the ECM (Takagi et al., 2003).
Within the central nervous system, most integrin isoforms are expressed throughout the brain with some degree of variation across different regions (Clegg et al., 2003). In addition to their roles in other cells, integrins are found to play a role in synapse regulation and plasticity within neurons (Park and Goda, 2016).
Integrin’s primary connection to the actin cytoskeleton is through the previously mentioned talin, which binds to the 𝛽-subunit cytoplasmic tail of the integrin dimer. Talin contains multiple binding sites for actin, which are adjacent to a vinculin binding site (Hemmings et al., 1996). Vinculin is a protein which in addition to binding talin, can also bind actin, strengthening the connection between the two (Rosowksi et al., 2018). This vinculin binding site is only exposed when talin undergoes deformation by being stretched open (Lee et al., 2007). This reinforces talin-actin interactions when under sufficient tension by causing a vinculin-mediated increase in talin-actin interactions following the exposure of new vinculin binding sites. This is supported by cells expressing vinculin exerting far greater forces than cells lacking vinculin (Mierke et al, 2007).
Another key player in cell-matrix interactions is focal adhesion kinase (FAK), a non-receptor tyrosine kinase, a signaling molecule activated by FAs and serves as a relay of information between FAs and the rest of the cell (Tapial Martínez et al., 2020). FAK is activated when the molecule forms a complex with the FA and is stretched open when tension is present (Seong et al., 2013). When activated FAK can form a complex with c-Src which is involved in multiple signaling pathways, most of which are related to cell motility and growth (Mitra and Schlaepfer, 2006).
While the binding patterns mentioned above seem to be the dominant interactions that lead to propper FA function, most FA proteins can bind with multiple other FA proteins. Although this would suggest that the proteins are clumped together somewhat haphazardly, FA structure is actually precisely regulated (Parsons et al., 2010). Part of this regulation is due to the spatial distribution of proteins with the FA, which produces functional subregion (Schwartz, 2011). Signals from the ECM will pass through the integrin signaling layer (ISL) and are then carried through the force transduction layer (FTL) before reaching the actin regulatory layer (ARL). This nano-organization has been shown to play an important role in regulating talin-mediated vinculin activation, and is likely to do the same for many other FA protein interactions (Case et al., 2015).
Biochemical Response to ECM Properties
Cells and the extracellular matrix engage in a bidirectional relationship in which the assembly of the ECM is regulated by signaling pathways within the cell, whose activity is dependent on cell surface receptors that recognize ECM proteins. This leads to variation in ECM biochemical composition, morphology, and rigidity, imparting chemical and physical qualities which facilitate proper tissue function (Gasiorowski et al., 2013). In this section I will explore some of these relationships between cells and the ECM.
It has long been known that the ECM plays a large role in determination of cell shape (Gospodarowicz et al., 1978). This interaction is similarly dependent on bidirectional signaling, in which the ECM provides contextual cues to the cells, thereby directing their phenotype, while cells remodel the ECM, altering the context itself (Gjorevski and Nelson, 2009). Changes in cell shape are largely due to the rigidity of the matrix, and therefore plays a large role in cell differentiation (Engler et al., 2006). Multiple ECM components have been shown to have a role in regulating branching morphogenesis in various cell types through changes in rigidity and by activating signaling cascades within the cell upon binding (Rozario and DeSimone, 2009). Other factors may affect this interaction. For example, in neurons, the response to ECM rigidity is regulated by neuron-astroglia interactions (Georges et al., 2007).
These effects are not limited to morphology and morphogenesis but extend also to migration, where the ECM plays a critical role in properly directing the cell (Yamada and Sixt, 2019). Before the cell even moves, the ECM directs cell polarity, an important factor in proper cell migration (Manninen, 2015). This is accompanied by an ECM dependent regulation of migration speed (Palecek et al., 1997).
In turn, cells direct a dynamic process of ECM degradation and remodeling which ultimately determines the properties of the ECM (Lu et al., 2011). One of the primary, and possibly simplest, mechanisms for ECM degradation is with the aid of proteinases which can selectively degrade a specified ECM protein (Cawston and Young, 2009). Alternatively, ECM proteins can often be regulated by posttranslational modifications (Frantz et al., 2010). A good example of this would be collagen, which can be cross-linked either covalently or noncovalently, causing a substantial change in ECM topography and rigidity (Levental et al., 2009). These processes of ECM degradation and regulation are themselves regulated either by gene regulation or environmentally (Page-McCaw et al., 2007).
Final Notes
These complex interactions between cells and the matrix highlights how cells do not exist as isolated systems within an organism. They are constantly exchanging information with their environment, which can be transmitted to other cells in the matrix, helping to add a foundation and network for communication and coordination between cells. It is therefore important to keep in mind the ECM when conducting research in any tissue, as to account for the behavior of cells in their native environment. This is equally true in neuroscience, where neurons often receive more attention than their neighboring glial cells. Expanding our understanding of the nervous system as more than a clump of neurons, but rather as a complex system of various cell types interconnected and supported by an ECM whose own composition may be as critical in proper cognitive function and development as the more popular neuronal circuits.
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