Susan E. LaFlamme

Expression of dominant-negative and chimeric subunits reveals an essential role for β1 integrin during myelination

Myelination represents a remarkable example of cell specialization and cell-cell interaction in development. During this process, axons are wrapped by concentric layers of cell membrane derived either from central nervous system (CNS) oligodendrocytes or peripheral nervous system Schwann cells. In the CNS, oligodendrocytes elaborate a membranous extension with an area of more than 1000 times that of the cell body. The mechanisms regulating this change in cell shape remain poorly understood. Signaling mechanisms regulated by cell surface adhesion receptors of the integrin family represent likely candidates. Integrins link the extracellular environment of the cell with both intracellular signaling molecules and the cytoskeleton and have been shown to regulate the activity of GTPases implicated in the control of cell shape. Our previous work has established that oligodendrocytes and their precursors express a limited repertoire of integrins. One of these, the alpha6beta1 laminin receptor, can interact with laminin-2 substrates to enhance oligodendrocyte myelin membrane formation in cell culture. However, these experiments do not address the important question of integrin function during myelination in vivo, nor do they define the respective roles of the alpha and beta subunits in the signaling pathways involved. Here, we use a dominant-negative approach to provide, for the first time, evidence that beta1 integrin function is required for myelination in vivo and use chimeric integrins to dissect apart the roles of the extracellular and cytoplasmic domains of the alpha6 subunit in the signaling pathways of myelination.

The Role of Conserved Amino Acid Motifs within the Integrin β3 Cytoplasmic Domain in Triggering Focal Adhesion Kinase Phosphorylation

Integrin-mediated adhesion of cells to extracellular matrix proteins triggers a variety of intracellular signaling pathways including a cascade of tyrosine phosphorylations. In many cell types, the cytoplasmic focal adhesion tyrosine kinase, FAK, appears to be the initial protein that becomes tyrosine-phosphorylated in response to adhesion; however, the molecular mechanisms regulating integrin-triggered FAK phosphorylation are not understood. Previous studies have shown that the integrin β1, β3, and β5 subunit cytoplasmic domains all contain sufficient information to trigger FAK phosphorylation when expressed in single-subunit chimeric receptors connected to an extracellular reporter. In the present study, β3 cytoplasmic domain deletion and substitution mutants were constructed to identify amino acids within the integrin β3 cytoplasmic domain that regulate its ability to trigger FAK phosphorylation. Cells transiently expressing chimeric receptors containing these mutant cytoplasmic domains were magnetically sorted and assayed for the tyrosine phosphorylation of FAK. Analysis of these mutants indicated that structural information in both the membrane-proximal and C-terminal segments of the β3 cytoplasmic domain is important for triggering FAK phosphorylation. In the C-terminal segment of the β3 cytoplasmic domain, the highly conserved NPXY motif was found to be required for the β3 cytoplasmic domain to trigger FAK phosphorylation. However, the putative FAK binding domain within the N-terminal segment of the β3 cytoplasmic domain was found to be neither required nor sufficient for this signaling event. We also demonstrate that the serine 752 to proline mutation, known to cause a variant of Glanzmanns thrombasthenia, inhibits the ability of the β3 cytoplasmic domain to signal FAK phosphorylation, suggesting that a single mutation in the β3 cytoplasmic domain can inhibit both “inside-out” and “outside-in” integrin signaling.

Perturbing integrin function inhibits microtubule growth from centrosomes, spindle assembly, and cytokinesis.

In many mammalian cell types, integrin-mediated cell-matrix adhesion is required for the G1–S transition of the cell cycle. As cells approach mitosis, a dramatic remodeling of their cytoskeleton accompanies dynamic changes in matrix adhesion, suggesting a mechanistic link. However, the role of integrins in cell division remains mostly unexplored. Using two cellular systems, we demonstrate that a point mutation in the β1 cytoplasmic domain (β1 tail) known to decrease integrin activity supports entry into mitosis but inhibits the assembly of a radial microtubule array focused at the centrosome during interphase, the formation of a bipolar spindle at mitosis and cytokinesis. These events are restored by externally activating the mutant integrin with specific antibodies. This is the first demonstration that the integrin β1 tail can regulate centrosome function, the assembly of the mitotic spindle, and cytokinesis.

INTEGRIN CYTOPLASMIC DOMAINS AS CONNECTORS TO THE CELL'S SIGNAL TRANSDUCTION APPARATUS

Integrins mediate the bidirectional transfer of signals across the plasma membrane. Integrin cytoplasmic domains provide one pathway linking integrin engagement with the cells signal transduction apparatus. Recent structure-function studies have defined regions of beta cytoplasmic domains required for integrin function and have identified distinct roles for individual alpha cytoplasmic domains in regulating cell behavior. Newly identified proteins that bind to integrin alpha and beta cytoplasmic domains have provided new insights and new questions into the mechanisms involved in integrin signaling.

The integrin β tail is required and sufficient to regulate adhesion signaling to Rac1

Rac1 is a small Rho family GTPase that regulates changes in cell morphology associated with cell spreading and migration. Integrin-mediated adhesion is known to activate Rac1 and to regulate the interaction of Rac1 with downstream effectors. Currently, it is not clear how integrins signal Rac1 activation following cell adhesion. Integrin β cytoplasmic domains (β-tails) are known to be required for integrin-mediated cell spreading, and isolated β tails expressed as tac-β tail chimeras can inhibit cell spreading indicating that protein interactions with β tails can regulate this process. Our recent studies demonstrated that the expression of constitutively activated Rac1 can restore cell spreading inhibited by tac β tail chimeras, suggesting a role for Rac1 in the regulation of cell spreading by β tails. Hence, we examined the role of β tails in integrin activation of Rac1. By using recombinant wild-type and mutant integrin heterodimers, we demonstrate that integrin β tails are required for adhesion to increase Rac1-GTP loading. We demonstrate that clustering tac-β tail chimeras, on the surface of cells in suspension, activates Rac1. Thus, β tails are not only required, but also sufficient for integrin-triggered Rac1 activation. Our findings indicate that integrin β-tails are an important link between integrin engagement and Rac1 signaling, and that protein interactions initiated at β tails are sufficient for integrins to regulate Rac1 activity.

The NPIY motif in the integrin β1 tail dictates the requirement for talin-1 in outside-in signaling

Protein interactions with the integrin β-subunit cytoplasmic domain (β-tail) are essential for adhesion-dependent processes, including cell spreading and the connection of integrins with actin filaments at adhesion sites. Talin-1 binds to the conserved membrane-proximal NPxY motif of β-tails (NPIY in β1 integrin) promoting the inside-out activation of integrins and providing a linkage between integrins and the actin cytoskeleton. Here, we characterize the role of interactions between talin-1 and β-tail downstream of integrin activation, in the context of recombinant integrins containing either the wild type (WT) or the (YA) mutant β1A tail, with a tyrosine to alanine substitution in the NPIY motif. In addition to inhibiting integrin activation, the YA mutation suppresses cell spreading, integrin signaling, focal adhesion and stress-fiber formation, as well as microtubule assembly. Constitutive activation of the mutant integrin restores these integrin-dependent processes, bringing into question the importance of the NPIY motif downstream of integrin activation. Depletion of talin-1 using TLN1 siRNA demonstrated that talin-1 is required for cell spreading, focal adhesion and stress-fiber formation, as well as microtubule assembly, even when cells are adhered by constitutively activated WT integrins. Depletion of talin-1 does not inhibit these processes when cells are adhered by constitutively activated mutant integrins, suggesting that the binding of an inhibitory protein to the NPIY motif negatively regulates integrin function when talin-1 is depleted. We identified filamin A (FLNa) as this inhibitory protein; it binds to the β1A tail in an NPIY-dependent manner and inhibition of FLNa expression in talin-1-depleted cells restores integrin function when cells are adhered by constitutively activated WT integrins. FLNa binds FilGAP, which is a negative regulator of Rac activation. Expression of the dominant inhibitory mutant, FilGAPΔGAP, which lacks GAP activity restores spreading in cells adhered by constitutively activated integrins containing the β1A tail, but not by integrins containing the β1D tail, which is known to bind poorly to FLNa. Together, these results suggest that the binding of talin-1 to the NPIY motif is required downstream of integrin activation to promote cell spreading by preventing the inappropriate recruitment of FLNa and FilGAP to the β1A tail. Our studies emphasize the importance of understanding the mechanisms that regulate the differential binding FLNa and talin-1 to the β1 tail downstream of integrin activation in promoting integrin function.

Integrins as regulators of the mitotic machinery

Mitotic spindle bipolarity defines a unique division plane that promotes the successful transmission of genetic material during cytokinesis. The positioning and orientation of the spindle determines the symmetry of cell division and the relative location of daughter cells, which regulate cell fate decisions that contribute to embryonic development and tissue differentiation. Recent studies have identified integrins as regulators of spindle positioning and orientation, as well as spindle bipolarity and cytokinesis. This review summarizes and discusses the current effort focused on understanding how integrins regulate these mitotic events.

Endothelial expression of the α6β4 integrin is negatively regulated during angiogenesis

Development and homeostasis of the vascular system requires integrin-facilitated cellular adhesion, migration, proliferation and survival. A specific role for the α6β4 integrin in the vasculature, however, has not been identified. Using immunohistochemistry, we observed α6β4 expression on the dermal microvasculature of human foreskin. Analysis of individual cells isolated from trypsin-disrupted foreskin tissue indicated that α6β4 was expressed by a subset of epithelial and endothelial cells, and not by smooth muscle cells. Expression of α6β4 was also analyzed during new vessel growth using explants of human saphenous vein cultured in fibrinogen gels. The results indicate that α6β4 is not expressed by outgrowing endothelial cells, and is downregulated by the original α6β4-positive endothelial cells of the explant. To determine whether α6β4 is expressed during angiogenesis in vivo, the expression of the β4 subunit was analyzed during the development of the mouse mystacial (whisker) pad. Immunohistochemical staining of the whisker pad indicates that β4 is expressed by the adult vasculature. To identify when and where β4 is turned on in the vasculature, we examined the whisker pads from the developing embryo (E19.5 pc), and from postnatal days zero (P0), three (P3) and seven (P7) pups. The expression of α6β4 was found to be turned on spatially and temporally from caudal to rostral regions and from the deep to superficial vasculature, correlating with the maturation of the whisker pad and its corresponding vasculature. Together, these findings suggest a potential role for α6β4 as a negative component of the angiogenic switch, whereas expression of α6β4 on the adult vasculature may indicate regions requiring additional adhesive mechanisms.

Integrins Regulate Microtubule Nucleating Activity of Centrosome through Mitogen-activated Protein Kinase/Extracellular Signal-regulated Kinase Kinase/Extracellular Signal-regulated Kinase (MEK/ERK) Signaling

Background: Centrosomal microtubule nucleation is important in the formation of the microtubule cytoskeleton. Results: An integrin mutant that suppresses MEK/ERK signaling substantially reduces microtubule nucleation. Expression of activated RAF-1 restores nucleation inhibited by the mutant integrin. Conclusion: Integrins promote microtubule nucleation by regulating MEK/ERK signaling. Significance: Environmental cues provided by cell-matrix adhesion contribute to the regulation of the microtubule nucleating activity of the centrosome. Microtubule nucleation is an essential step in the formation of the microtubule cytoskeleton. We recently showed that androgen and Src promote microtubule nucleation and γ-tubulin accumulation at the centrosome. Here, we explore the mechanisms by which androgen and Src regulate these processes and ask whether integrins play a role. We perturb integrin function by a tyrosine-to-alanine substitution in membrane-proximal NPIY motif in the integrin β1 tail and show that this mutant substantially decreases microtubule nucleation and γ-tubulin accumulation at the centrosome. Because androgen stimulation promotes the interaction of the androgen receptor with Src, resulting in PI3K/AKT and MEK/ERK signaling, we asked whether these pathways are inhibited by the mutant integrin and whether they regulate microtubule nucleation. Our results indicate that the formation of the androgen receptor-Src complex and the activation of downstream pathways are significantly suppressed when cells are adhered by the mutant integrin. Inhibitor studies indicate that microtubule nucleation requires MEK/ERK but not PI3K/AKT signaling. Importantly, the expression of activated RAF-1 is sufficient to rescue microtubule nucleation inhibited by the mutant integrin by promoting the centrosomal accumulation of γ-tubulin. Our data define a novel paradigm of integrin signaling, where integrins regulate microtubule nucleation by promoting the formation of androgen receptor-Src signaling complexes to activate the MEK/ERK signaling pathway.

Androgen and Src signaling regulate centrosome activity

Microtubules nucleated from γ-tubulin ring complexes located at the centrosome regulate the localization of organelles, promote vesicular transport and direct cell migration. Although several signaling mechanisms have been identified that regulate microtubule dynamics during interphase, signaling pathways that promote microtubule nucleation remain elusive. We assayed microtubule regrowth following nocodazole washout in human fibroblasts and CHO-K1 cells adhered to fibronectin in either normal serum-free medium or the serum-free, growth-promoting medium, CCM1, which contains IGF1 and androgen, as well as other nutrients. The results indicate that integrin-mediated adhesion is not sufficient to promote rapid microtubule regrowth in either cell type. The addition of androgen, but not IGF1, for 5 minutes was sufficient to promote rapid regrowth and this occurred by a mechanism requiring the androgen receptor. Since Src is a component of the cytoplasmic androgen-receptor-signaling complex, we examined its role using Src siRNA, the Src kinase inhibitor SU6656, and the expression of a constitutively active Src mutant. The data show that Src signaling is both required and sufficient to promote rapid microtubule regrowth in cells adhered to fibronectin. Measurement of the density of microtubules close to the centrosome and the rates of GFP-EB1-labeled microtubules emanating from the centrosome indicated that Src signaling promotes microtubule nucleation. Furthermore, recovery of GFP–γ-tubulin at the centrosome following photobleaching and measurements of endogenous γ-tubulin levels at the centrosome showed that androgen and Src signaling regulate the levels of centrosomal γ-tubulin. Thus, we propose that androgen and Src signaling regulate microtubule nucleation during interphase by promoting the centrosomal localization of γ-tubulin.