Matrix regulators in neural stem cell functions

Background

Neural stem/progenitor cells (NSPCs) reside within a complex and dynamic extracellular microenvironment, or niche. This niche regulates fundamental aspects of their behavior during normal neural development and repair. Precise yet dynamic regulation of NSPC self-renewal, migration, and differentiation is critical and must persist over the life of an organism.

Scope of review

In this review, we summarize some of the major components of the NSPC niche and provide examples of how cues from the extracellular matrix regulate NSPC behaviors. We use proteoglycans to illustrate the many diverse roles of the niche in providing temporal and spatial regulation of cellular behavior.

Major conclusions

The NSPC niche is comprised of multiple components that include; soluble ligands, such as growth factors, morphogens, chemokines, and neurotransmitters, the extracellular matrix, and cellular components. As illustrated by proteoglycans, a major component of the extracellular matrix, the NSPC, niche provides temporal and spatial regulation of NSPC behaviors.

General significance

The factors that control NSPC behavior are vital to understand as we attempt to modulate normal neural development and repair. Furthermore, an improved understanding of how these factors regulate cell proliferation, migration, and differentiation, crucial for malignancy, may reveal novel anti-tumor strategies. This article is part of a Special Issue entitled Matrix-mediated cell behaviour and properties.     

Extracellular matrix interactions 2: Extracellular matrix structure is important for growth factor localization and function

Summary The influence of extracellular matrix components and of extracellular matrix structure on in vitro cell growth was investigated in the UWOV2 (Pf), protein-free cell culture model. This cell line constitutively produces an ordered extracellular matrix in the absence of any exogenous protein or growth factor. Extracellular matrix from UWOV2 (Pf) cells was found to contain both transforming growth factor β (TGFβ) and platelet-derived growth factor (PDGF), which were shown to have an autostimulatory role for UWOV2 (Pf) cell growth. Matrix structure was shown to be important for allowing expression of the functional activity of these two growth factors. In addition, a nonuniform distribution of PDGF, embedded within the matrix structure, was demonstrated by immunoelectronmicroscopy. Apart from these two well-defined growth factors, additional but as yet unidentified growth stimulatory factor(s) were extractable from UWOV2 (Pf) extracellular matrix. These investigations indicate the potential role of extracellular matrix both as a mechanism for concentrating as well as modulating the function of cellular growth factors.     

A genetic strategy for the dynamic and graded control of cell mechanics, motility, and matrix remodeling

Cellular mechanical properties have emerged as central regulators of many critical cell behaviors, including proliferation, motility, and differentiation. Although investigators have developed numerous techniques to influence these properties indirectly by engineering the extracellular matrix (ECM), relatively few tools are available to directly engineer the cells themselves. Here we present a genetic strategy for obtaining graded, dynamic control over cellular mechanical properties by regulating the expression of mutant mechanotransductive proteins from a single copy of a gene placed under a repressible promoter. With the use of constitutively active mutants of RhoA GTPase and myosin light chain kinase, we show that varying the expression level of either protein produces graded changes in stress fiber assembly, traction force generation, cellular stiffness, and migration speed. Using this approach, we demonstrate that soft ECMs render cells maximally sensitive to changes in RhoA activity, and that by modulating the ability of cells to engage and contract soft ECMs, we can dynamically control cell spreading, migration, and matrix remodeling. Thus, in addition to providing quantitative relationships between mechanotransductive signaling, cellular mechanical properties, and dynamic cell behaviors, this strategy enables us to control the physical interactions between cells and the ECM and thereby dictate how cells respond to matrix properties.     

Syndecan, a developmentally regulated cell surface proteoglycan that binds extracellular matrix and growth factors

Cellular behaviour during development is dictated, in part, by the insoluble extracellular matrix and the soluble growth factor peptides, the major molecules responsible for integrating cells into morphologically and functionally defined groups. These extracellular molecules influence cellular behaviour by binding at the cell surface to specific receptors that transduce intracellular signals in various ways not yet fully clear. Syndecan, a cell surface proteoglycan found predominantly on epithelia in mature tissues binds both extracellular matrix components (fibronectin, collagens I, III, V, and thrombospondin) and basic fibroblast growth factor (bFGF). Syndecan consists of chondroitin sulfate and heparan sulphate chains linked to a 31 kilodalton (kDa) integral membrane protein. Syndecan represents a family of integral membrane proteoglycans that differ in extracellular domains, but share cytoplasmic domains. Syndecan behaves as a matrix receptor: it binds selectively to components of the extracellular matrix, associates intracellularly with the actin cytoskeleton when cross-linked at the cell surface, its extracellular domain is shed upon cell rounding and it localizes solely to basolateral surfaces of simple epithelia. Mammary epithelial cells made syndecan-deficient become fibroblastic in morphology and cell behaviour, showing that syndecan maintains epithelial cell morphology. Syndecan changes in quantity, location and structure during development: it appears initially on four-cell embryos (prior to its known matrix ligands), becomes restricted in the pre-implementation embryo to the cells that will form the embryo proper, changes its expression due to epithelial-mesenchymal interactions (for example, induced in kidney mesenchyme by the ureteric bud), and with association of cells with extracellular matrix (for example, during B-cell differentiation), and ultimately, in mature tissues becomes restricted to epithelial tissues. The number and size of its glycosaminoglycan chains vary with changes in cell shape and organization yielding tissue type-specific polymorphic forms of syndecan. Its interactions with the major extracellular effector molecules that influence cell behaviour, its role in maintaining cell shape and its spatial and temporal changes in expression during development indicate that syndecan is involved in morphogenesis.     

The mouse pubic symphysis as a remodeling system: morphometrical analysis of proliferation and cell death during pregnancy, partus and postpartum

Marked changes in mice pubic symphysis occur by the end of pregnancy. Tissue remodeling involves a dynamic balance between cell proliferation and programmed cell death as well as changes in the extracellular matrix components. Therefore, it is important to consider both of these cellular behaviors when investigating the mechanism that regulates interpubic tissue remodeling, growth during late pregnancy and partus ensuring involution during the postpartum period. Proliferating and programmed death cells were identified by immunohistochemistry (proliferating cell nuclear antigen and TUNEL detection, respectively) and the rates at which these processes occurred were determined by morphometric analysis. The results demonstrated that cellular proliferation was intense during the period of ligament formation, from D15 to D18, thereafter abruptly declining on D19. From parturition (D19) onwards, an ever-increasing decline in the cellular proliferation levels could be observed. The quantitative analyses of cellular death showed opposite results when compared to cellular proliferation. During early pregnancy the cycle of cellular renovation was clearly proliferative and during late mouse pregnancy the cycle was directed by programmed cellular death. Although the high levels of cellular death during postpartum involution could be shown by the TUNEL-positive cells, we were unable to observed picnotic nucleus at the light microscopy.     

Modelling cell migration strategies in the extracellular matrix

The extracellular matrix (ECM) is a highly organised structure with the capacity to direct cell migration through their tendency to follow matrix fibres, a process known as contact guidance. Amoeboid cell populations migrate in the ECM by making frequent shape changes and have minimal impact on its structure. Mesenchymal cells actively remodel the matrix to generate the space in which they can move. In this paper, these different types of movement are studied through simulation of a continuous transport model. It is shown that the process of contact guidance in a structured ECM can spatially organise cell populations. Furthermore, when combined with ECM remodelling, it can give rise to cellular pattern formation in the form of "cell-chains" or networks without additional environmental cues such as chemoattractants. These results are applied to a simple model for tumour invasion where it is shown that the interactions between invading cells and the ECM structure surrounding the tumour can have a profound impact on the pattern and rate of cell infiltration, including the formation of characteristic "fingering" patterns. The results are further discussed in the context of a variety of relevant processes during embryonic and adult stages.     

The role of proteoglycans in cell adhesion, migration and proliferation.

Proteoglycans comprise a part of the extracellular matrix that participates in the molecular events that regulate cell adhesion, migration and proliferation. Their structural diversity and tissue distribution suggest a functional versatility not generally encountered for other extracellular matrix components. This versatility is mainly dictated by their molecular interactions and their ability to regulate the activity of key molecules involved in several biological events. This molecular cooperativity either promotes or inhibits cell adhesion, migration and proliferation. A growing number of studies indicate that proteoglycans can play a direct role in these cellular events by functioning either as receptors or as ligands for molecules that are required for these events to occur. Such studies support a role for proteoglycans as important effectors of cellular processes that constitute the basis of development and disease.     

Gelatinase A activity directly modulates melanoma cell adhesion and spreading.

Interaction of cells with the extracellular matrix (ECM) plays an important role in the regulation of cell behavior. Formation of adhesive contacts leads to transduction of signals into the cell and results in altered gene expression and modulation of the cellular phenotype. Specific adhesive interactions of the fibronectin and vitronectin receptors with their ligands in the matrix modulates expression of ECM-degrading metalloproteases. These proteases are involved in the acquisition of the invasive phenotype by a number of cell types. The activity of matrix metalloproteases (MMPs) is reduced by endogenous inhibitors referred to as tissue inhibitors of metalloproteases (TIMPs). Alterations in the balance between the activity of MMPs and TIMPs alters cellular invasion through effects on matrix degradation. In this study we demonstrate that inhibition of endogenous gelatinase A activity in A2058 human melanoma cells results in enhanced cellular adhesion. To further explore this phenomenon, we have used retroviral infection vectors to control the amount of the MMP inhibitor TIMP-2 in human melanoma A2058 cells. Altering the production of TIMP-2 modulates not only proteolysis of the extracellular matrix, but also the adhesive and spreading properties of the cells and results in altered cell morphology. These effects of TIMP-2 appear to be mediated by inhibition of gelatinase A activity. We conclude that gelatinase A, in addition to contributing to proteolysis of ECM components, also functions to proteolyse cell surface components that mediate attachment of A2058 cells to the ECM. Thus, gelatinase A may function to modulate cell attachment and facilitate cell migration and invasion.     

A transitional extracellular matrix instructs cell behavior during muscle regeneration

Urodele amphibians regenerate appendages through the recruitment of progenitor cells into a blastema that rebuilds the lost tissue. Blastemal formation is accompanied by extensive remodeling of the extracellular matrix. Although this remodeling process is important for appendage regeneration, it is not known whether the remodeled matrix directly influences the generation and behavior of blastemal progenitor cells. By integrating in vivo 3-dimensional spatiotemporal matrix maps with in vitro functional time-lapse imaging, we show that key components of this dynamic matrix, hyaluronic acid, tenascin-C and fibronectin, differentially direct cellular behaviors including DNA synthesis, migration, myotube fragmentation and myoblast fusion. These data indicate that both satellite cells and fragmenting myofibers contribute to the regeneration blastema and that the local extracellular environment provides instructive cues for the regenerative process. The fact that amphibian and mammalian myoblasts exhibit similar responses to various matrices suggests that the ability to sense and respond to regenerative signals is evolutionarily conserved.     

Role of the Z band in the mechanical properties of the heart

In striated muscle the mechanism of contraction involves the cooperative movement of contractile and elastic components. This review emphasizes a structural approach that describes the cellular and extracellular components with known anatomical, biochemical, and physical properties that make them candidates for these contractile and elastic components. Classical models of contractile and elastic elements and their underlying assumptions are presented. Mechanical properties of cardiac and skeletal muscle are compared and contrasted and then related to ultrastructure. Information from these approaches leads to the conclusion that the Z band is essential for muscle contraction. Our review of Z band structure shows the Z band at the interface where extracellular components meet the cell surface. The Z band is also the interface from cell surface to myofibril, from extra-myofibrillar to myofibril, and finally from sarcomere to sarcomere. Our studies of Z band in defined physiologic states show that this lattice is an integral part of the contractile elements and can function as an elastic component. The Z band is a complex dynamic lattice uniquely suited to play several roles in muscle contraction.     

Cell cycle dependent TN-C promoter activity determined by live cell imaging

The extracellular matrix protein tenascin-C plays a critical role in development, wound healing, and cancer progression, but how it is controlled and how it exerts its physiological responses remain unclear. By quantifying the behavior of live cells with phase contrast and fluorescence microscopy, the dynamic regulation of TN-C promoter activity is examined. We employ an NIH 3T3 cell line stably transfected with the TN-C promoter ligated to the gene sequence for destabilized green fluorescent protein (GFP). Fully automated image analysis routines, validated by comparison with data derived from manual segmentation and tracking of single cells, are used to quantify changes in the cellular GFP in hundreds of individual cells throughout their cell cycle during live cell imaging experiments lasting 62 h. We find that individual cells vary substantially in their expression patterns over the cell cycle, but that on average TN-C promoter activity increases during the last 40% of the cell cycle. We also find that the increase in promoter activity is proportional to the activity earlier in the cell cycle. This work illustrates the application of live cell microscopy and automated image analysis of a promoter-driven GFP reporter cell line to identify subtle gene regulatory mechanisms that are difficult to uncover using population averaged measurements.     

Structure And Evolution Of Calcium-Modulated Protein

Abstract

The question posed by the science of analytical histology is how the properties and interactions of the components of the tissues determine their organization in the organs. The relevant components of the tissues are the cells and the extracellular matrix. The ability of cohering populations of cells to self-assemble structured tissues by cell sorting out offers an important opportunity for the experimental study of the mechanisms by which the cells and extracellular matrix interact to determine structure. The investigator can manipulate the initial organization and the cellular composition of the system and, in favorable situations, the composition of the extracellular matrix and the activities of candidate adhesive molecules.     

The ECM moves during primitive streak formation--computation of ECM versus cellular motion

Galileo described the concept of motion relativity--motion with respect to a reference frame--in 1632. He noted that a person below deck would be unable to discern whether the boat was moving. Embryologists, while recognizing that embryonic tissues undergo large-scale deformations, have failed to account for relative motion when analyzing cell motility data. A century of scientific articles has advanced the concept that embryonic cells move ("migrate") in an autonomous fashion such that, as time progresses, the cells and their progeny assemble an embryo. In sharp contrast, the motion of the surrounding extracellular matrix scaffold has been largely ignored/overlooked. We developed computational/optical methods that measure the extent embryonic cells move relative to the extracellular matrix. Our time-lapse data show that epiblastic cells largely move in concert with a sub-epiblastic extracellular matrix during stages 2 and 3 in primitive streak quail embryos. In other words, there is little cellular motion relative to the extracellular matrix scaffold--both components move together as a tissue. The extracellular matrix displacements exhibit bilateral vortical motion, convergence to the midline, and extension along the presumptive vertebral axis--all patterns previously attributed solely to cellular "migration." Our time-resolved data pose new challenges for understanding how extracellular chemical (morphogen) gradients, widely hypothesized to guide cellular trajectories at early gastrulation stages, are maintained in this dynamic extracellular environment. We conclude that models describing primitive streak cellular guidance mechanisms must be able to account for sub-epiblastic extracellular matrix displacements.     

Brushes,cables, and anchors: Recent insights into multiscale assembly and mechanics of cellular structural networks

The remarkable ability of living cells to sense, process, and respond to mechanical stimuli in their environment depends on the rapid and efficient interconversion of mechanical and chemical energy at specific times and places within the cell. For example, application of force to cells leads to conformational changes in specific mechanosensitive molecules which then trigger cellular signaling cascades that may alter cellular structure, mechanics, and migration and profoundly influence gene expression. Similarly, the sensitivity of cells to mechanical stresses is governed by the composition, architecture, and mechanics of the cellular cytoskeleton and extracellular matrix (ECM), which are in turn driven by molecular-scale forces between the constituent biopolymers. Understanding how these mechanochemical systems coordinate over multiple length and time scales to produce orchestrated cell behaviors represents a fundamental challenge in cell biology. Here, we review recent advances in our understanding of these complex processes in three experimental systems: the assembly of axonal neurofilaments, generation of tensile forces by actomyosin stress fiber bundles, and mechanical control of adhesion assembly.     

Fibroblasts retain their tissue phenotype when grown in three-dimensional collagen gels

Fibroblasts are responsible for the synthesis, assembly, deposition, and organization of extracellular matrix molecules, and thus determine the morphology of connective tissues. Deposition of matrix molecules occurs in extracellular compartments, where the sequential stages are under cellular control. Cell orientation/polarity is important in determining how the cell orients these extracytoplasmic compartments and therefore how the matrix is assembled and oriented. However, the control of cell orientation is not understood. Fibroblasts from three tissues with different morphologies were studied to determine whether cells maintained their characteristic phenotype. Fibroblasts from cornea, which in vivo are oriented in orthogonal layers along with their matrix; from tendon, a uniaxial connective tissue, where cells orient parallel to each other; and from dermis, a connective tissue with no apparent cellular orientation, were used to study cell morphology and orientation in three-dimensional collagen gels. The different cells were grown for 3 and 7 days in identical three-dimensional collagen gels with a nonoriented matrix. Confocal fluorescence microscopy demonstrated that corneal fibroblasts oriented perpendicular to one another at 3 days, and after 7 days in hydrated gels these cells formed orthogonal sheets. Tendon fibroblasts were shown by the same methods to orient parallel to one another in bundles at both 3 and 7 days, throughout the depth of the gel. Dermal fibroblasts showed no apparent orientation throughout the hydrated gels at either time point examined. The organization of these different cell types was consistent with their tissue of origin as was the cell structure and polarity. These studies imply that cellular and tissue phenotype is innate to differentiated fibroblasts and that these cells will orient in a tissue-specific manner regardless of the extracellular matrix present.     

The membrane form of the DNA repair protein Ku interacts at the cell surface with metalloproteinase 9

The Ku heterodimer (Ku70/Ku80) plays a central role in DNA double-strand breaks repair. Ku is also expressed on the cell surface of different types of cells where its function remains poorly understood. From a yeast two-hybrid screen, we have identified a specific interaction between the core region of Ku80 and the hemopexin domain of metalloproteinase 9 (MMP-9), a key enzyme involved in the degradation of extracellular matrix (ECM) components. Ku associates with MMP-9 on the surface of leukemic cells as demonstrated by co-immunoprecipitation experiments in membrane extracts and double-label immunofluorescence studies. In normal and tumoral migratory cells, Ku80 and MMP-9 colocalize at the periphery of leading edge of cells and cellular invasion of collagen IV matrices was blocked by antibodies directed against Ku70 or Ku80 subunits as well as by Ku80-specific antisense oligonucleotides. Our results indicate that Ku and MMP-9 interact at the cell membrane of highly invasive hematopoietic cells of normal and tumoral origin and document the unexpected importance of the membrane-associated form of Ku in the regulation of ECM remodelling.