The Rho family GEF Asef2 regulates cell migration in three dimensional (3D) collagen matrices through myosin II.

Cell migration is fundamental to a variety of physiological processes, including tissue development, homeostasis, and regeneration. Migration has been extensively studied with cells on 2-dimensional (2D) substrates, but much less is known about cell migration in 3D environments. Tissues and organs are 3D, which is the native environment of cells in vivo, pointing to a need to understand migration and the mechanisms that regulate it in 3D environments. To investigate cell migration in 3D environments, we developed microfluidic devices that afford a controlled, reproducible platform for generating 3D matrices. Using these devices, we show that the Rho family guanine nucleotide exchange factor (GEF) Asef2 inhibits cell migration in 3D type I collagen (collagen I) matrices. Treatment of cells with the myosin II (MyoII) inhibitor blebbistatin abolished the decrease in migration by Asef2. Moreover, Asef2 enhanced MyoII activity as shown by increased phosphorylation of serine 19 (S19). Furthermore, Asef2 increased activation of Rac, which is a Rho family small GTPase, in 3D collagen I matrices. Inhibition of Rac activity by treatment with the Rac-specific inhibitor NSC23766 abrogated the Asef2-promoted increase in S19 MyoII phosphorylation. Thus, our results indicate that Asef2 regulates cell migration in 3D collagen I matrices through a Rac-MyoII-dependent mechanism.

Activation of Rac by Asef2 promotes myosin II-dependent contractility to inhibit cell migration on type I collagen.

Non-muscle myosin II (MyoII) contractility is central to the regulation of numerous cellular processes, including migration. Rho is a well-characterized modulator of actomyosin contractility, but the function of other GTPases, such as Rac, in regulating contractility is currently not well understood. Here, we show that activation of Rac by the guanine nucleotide exchange factor Asef2 (also known as SPATA13) impairs migration on type I collagen through a MyoII-dependent mechanism that enhances contractility. Knockdown of endogenous Rac or treatment of cells with a Rac-specific inhibitor decreases the amount of active MyoII, as determined by serine 19 (S19) phosphorylation, and negates the Asef2-promoted increase in contractility. Moreover, treatment of cells with blebbistatin, which inhibits MyoII activity, abolishes the Asef2-mediated effect on migration. In addition, Asef2 slows the turnover of adhesions in protrusive regions of cells by promoting large mature adhesions, which has been linked to actomyosin contractility, with increased amounts of active ß1 integrin. Hence, our data reveal a new role for Rac activation, promoted by Asef2, in modulating actomyosin contractility, which is important for regulating cell migration and adhesion dynamics.

Glia co-culture with neurons in microfluidic platforms promotes the formation and stabilization of synaptic contacts.

Two novel microfluidic cell culture schemes, a vertically-layered set-up and a four chamber set-up, were developed for co-culturing central nervous system (CNS) neurons and glia. The cell chambers in these devices were separated by pressure-enabled valve barriers, which permitted us to control communication between the two cell types. The unique design of these devices facilitated the co-culture of glia with neurons in close proximity (∼50-100 µm), differential transfection of neuronal populations, and dynamic visualization of neuronal interactions, such as the development of synapses. With these co-culture devices, initial synaptic contact between neurons transfected with different fluorescent markers, such as green fluorescent protein (GFP) and mCherry-synaptophysin, was imaged using high-resolution fluorescence microscopy. The presence of glial cells had a profound influence on synapses by increasing the number and stability of synaptic contacts. Interestingly, as determined by liquid chromatography-ion mobility-mass spectrometry, neuron-glia co-cultures produced elevated levels of soluble factors compared to that secreted by individual neuron or glia cultures, suggesting a potential mechanism by which neuron-glia interactions could modulate synaptic function. Collectively, these results show that communication between neurons and glia is critical for the formation and stability of synapses and point to the importance of developing neuron-glia co-culture systems such as the microfluidic platforms described in this study.

The endosomal adaptor protein APPL1 impairs the turnover of leading edge adhesions to regulate cell migration.

Cell migration is a complex process that requires the integration of signaling events that occur in distinct locations within the cell. Adaptor proteins, which can localize to different subcellular compartments, where they bring together key signaling proteins, are emerging as attractive candidates for controlling spatially coordinated processes. However, their function in regulating cell migration is not well understood. In this study, we demonstrate a novel role for the adaptor protein containing a pleckstrin-homology (PH) domain, phosphotyrosine-binding (PTB) domain, and leucine zipper motif 1 (APPL1) in regulating cell migration. APPL1 impairs migration by hindering the turnover of adhesions at the leading edge of cells. The mechanism by which APPL1 regulates migration and adhesion dynamics is by inhibiting the activity of the serine/threonine kinase Akt at the cell edge and within adhesions. In addition, APPL1 significantly decreases the tyrosine phosphorylation of Akt by the nonreceptor tyrosine kinase Src, which is critical for Akt-mediated cell migration. Thus, our results demonstrate an important new function for APPL1 in regulating cell migration and adhesion turnover through a mechanism that depends on Src and Akt. Moreover, our data further underscore the importance of adaptor proteins in modulating the flow of information through signaling pathways.

A versatile valve-enabled microfluidic cell co-culture platform and demonstration of its applications to neurobiology and cancer biology.

A versatile microfluidic platform allowing co-culture of multiple cell populations in close proximity with separate control of their microenvironments would be extremely valuable for many biological applications. Here, we report a simple and compact microfluidic platform that has these desirable features and allows for real-time, live-cell imaging of cell-cell interactions. Using a pneumatically/hydraulically controlled poly(dimethylsiloxane) (PDMS) valve barrier, distinct cell types can be cultured in side-by-side microfluidic chambers with their optimum culture media and treated separately without affecting the other cell population. The platform is capable of both two-dimensional and three-dimensional cell co-culture and through variations of the valve barrier design, the platform allows for cell-cell interactions through either direct cell contact or soluble factors alone. The platform has been used to perform dynamic imaging of synapse formation in hippocampal neurons by separate transfection of two groups of neurons with fluorescent pre- and post-synaptic protein markers. In addition, cross-migration of 4T1 tumor cells and endothelial cells has been studied under normoxic and hypoxic conditions, which revealed different migration patterns, suggesting the importance of the microenvironments in cell-cell interactions and biological activities.

An APPL1/Akt signaling complex regulates dendritic spine and synapse formation in hippocampal neurons.

The formation and plasticity of dendritic spines and synapses, which are poorly understood on a molecular level, are critical for cognitive functions, such as learning and memory. The adaptor protein containing a PH domain, PTB domain, and leucine zipper motif (APPL1) is emerging as a critical regulator of various cellular processes in non-neuronal cells, but its function in the nervous system is not well understood. Here, we show that APPL1 localizes to dendritic spines and synapses and regulates the development of these structures in hippocampal neurons. Knockdown of endogenous APPL1 using siRNA led to a significant decrease in the number of spines as well as synapses and this defect could be rescued by expression of siRNA-resistant APPL1. Expression of exogenous APPL1 increased the spine and synaptic density and the amount of surface GluR1-containing α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid receptors (AMPARs). Deletion of the C-terminal phosphotyrosine binding domain of APPL1, which binds the serine/threonine kinase Akt, resulted in a significant decrease in the spine and synaptic density, suggesting a role for Akt in regulating the development of these structures. Consistent with this, knockdown of Akt with siRNA or expression of dominant negative Akt led to a dramatic decrease in spine and synapse formation. In addition, APPL1 increased the amount of active Akt in spines and synapses and the effects of APPL1 on these structures were dependent on Akt, indicating that Akt is an effector of APPL1 in the regulation of these processes. Moreover, APPL1 signaling modulates spine and synapse formation through p21-activated kinase (PAK). Thus, our results indicate that APPL1 signaling through Akt and PAK is critical for spine and synaptic development and point to a role for APPL1 and its effectors in regulating cognitive function.

Co-culture of neurons and glia in a novel microfluidic platform.

In this study, we developed a microfluidic cell co-culture platform that permits individual manipulation of the microenvironment of different cell types. Separation of the cell culture chambers is controlled by changing the position of a microfabricated valve, which serves as a barrier between the chambers. This unique feature of our platform allowed us to maintain healthy co-cultures of hippocampal neurons and glia for several weeks under optimal conditions. Controlled fluidic exchange between the cell culture chambers provided neurons with a continuous supply of in situ conditioned glia media that was critical for their survival. Using the barrier valve, we transfected neurons in the adjacent chambers with green fluorescent protein (GFP) and mCherry cDNA, respectively, with a transfection efficiency of approximately 40%. Co-culture with glia further enhanced the transfection efficiency of neurons to almost 60%. Thus the microfluidic devices offer a novel platform for the long-term culture, transfection, and individual treatment of central nervous system cells.

The Rho-family GEF Asef2 activates Rac to modulate adhesion and actin dynamics and thereby regulate cell migration.

Asef2 is a recently identified Rho-family guanine nucleotide exchange factor (GEF) that has been implicated in the modulation of actin, but its function in cell migration and adhesion dynamics is not well understood. In this study, we show that Asef2 is an important regulator of cell migration and adhesion assembly and disassembly (turnover). Asef2 localizes with actin at the leading edge of cells. Knockdown of endogenous Asef2 impairs migration and significantly slows the turnover of adhesions. Asef2 enhances both Rac1 and Cdc42 activity in HT1080 cells, but only Rac1 is crucial for the Asef2-promoted increase in migration and adhesion turnover. Phosphoinositide 3-kinase (PI3K) and the serine/threonine kinase Akt are also essential for the Asef2-mediated effects on migration and adhesion turnover. Consistent with this, Asef2 increases the amount of active Akt at the leading edge of cells. Asef2 signaling leads to an overall decrease in Rho activity, which is crucial for stimulating migration and adhesion dynamics. Thus, our results reveal an important new role for Asef2 in promoting cell migration and rapid adhesion turnover by coordinately regulating the activities of Rho-family GTPases.

N-wasp and the arp2/3 complex are critical regulators of actin in the development of dendritic spines and synapses.

Changes in the number, size, and shape of dendritic spines are associated with synaptic plasticity, which underlies cognitive functions such as learning and memory. This plasticity is attributed to reorganization of actin, but the molecular signals that regulate this process are poorly understood. In this study, we show neural Wiskott-Aldrich syndrome protein (N-WASP) regulates the formation of dendritic spines and synapses in hippocampal neurons. N-WASP localized to spines and active, functional synapses as shown by loading with FM4-64 dye. Knock down of endogenous N-WASP expression by RNA interference or inhibition of its activity by treatment with a specific inhibitor, wiskostatin, caused a significant decrease in the number of spines and excitatory synapses. Deletion of the C-terminal VCA region of N-WASP, which binds and activates the actin-related protein 2/3 (Arp2/3) complex, dramatically decreased the number of spines and synapses, suggesting activation of the Arp2/3 complex is critical for spine and synapse formation. Consistent with this, Arp3, like N-WASP, was enriched in spines and excitatory synapses and knock down of Arp3 expression impaired spine and synapse formation. A similar defect in spine and synapse formation was observed when expression of an N-WASP activator, Cdc42, was knocked down. Thus, activation of N-WASP and, subsequently, the Arp2/3 complex appears to be an important molecular signal for regulating spines and synapses. Arp2/3-mediated branching of actin could be a mechanism by which dendritic spine heads enlarge and subsequently mature. Collectively, our results point to a critical role for N-WASP and the Arp2/3 complex in spine and synapse formation.

alpha5 integrin signaling regulates the formation of spines and synapses in hippocampal neurons.

The actin-based dynamics of dendritic spines play a key role in synaptic plasticity, which underlies learning and memory. Although it is becoming increasingly clear that modulation of actin is critical for spine dynamics, the upstream molecular signals that regulate the formation and plasticity of spines are poorly understood. In non-neuronal cells, integrins are critical modulators of the actin cytoskeleton, but their function in the nervous system is not well characterized. Here we show that alpha5 integrin regulates spine morphogenesis and synapse formation in hippocampal neurons. Knockdown of alpha5 integrin expression using small interfering RNA decreased the number of dendritic protrusions, spines, and synapses. Expression of constitutively active or dominant negative alpha5 integrin also resulted in alterations in the number of dendritic protrusions, spines, and synapses. alpha5 integrin signaling regulates spine morphogenesis and synapse formation by a mechanism that is dependent on Src kinase, Rac, and the signaling adaptor GIT1. Alterations in the activity or localization of these molecules result in a significant decrease in the number of spines and synapses. Thus, our results point to a critical role for integrin signaling in regulating the formation of dendritic spines and synapses in hippocampal neurons.

8-OH-DPAT attenuates isoproterenol- but not forskolin-stimulated accumulation of cAMP in mediobasal hypothalamus.

Ovariectomized female rats were used to test the possibility that the 5-HT(1A) receptor agonist, 8-hydroxy-2-(di-N-propylamino) tetralin (8-OH-DPAT), inhibits cyclic AMP (cAMP) accumulation in the mediobasal hypothalamus. Tissue slices were incubated with forskolin or with the beta-adrenergic receptor agonist, isoproterenol, to stimulate accumulation of cAMP. Both compounds increased accumulation of cAMP. The 5-HT(1A) receptor agonist, 8-OH-DPAT, reduced cAMP accumulation after stimulation by isoproterenol, but not after forskolin stimulation. These findings are discussed in terms of putative differences in the mechanisms whereby 5-HT(1A) receptors are able to inhibit stimulation of adenylate cyclase. The potential significance of these findings to 5-HT(1A) receptor-mediated inhibition of female rat lordosis behavior is also discussed.

Restraint accentuates the effects of 5-HT2 receptor antagonists and a 5-HT1A receptor agonist on lordosis behavior.

The effect of restraint on lordosis behavior was examined in proestrous and ovariectomized, hormone-primed rats. Restraint durations from 5 to 60 min had no effect on lordosis behavior of proestrous rats. There was also no effect of 5 min restraint on lordosis behavior of ovariectomized rats hormonally primed with 10 microg estradiol benzoate and 500 microg progesterone. However, after intraperitoneal treatment with 1.0 mg/kg ketanserin tartrate (ketanserin), 5 min of restraint significantly reduced lordosis behavior of both groups of rats. The 5-min restraint combined with 0.50 or 0.75 mg/kg ketanserin reduced lordosis to mount (L/M) ratios of ovariectomized rats, while L/M ratios of proestrous rats were inhibited only by the 1.0 mg/kg dose. Increasing the restraint duration (10 or 15 min) reduced the dose of ketanserin necessary to reduce the L/M ratios of proestrous rats. Treatment with the selective serotonin (5-HT)(2C) receptor antagonist, SB206553 (2.5 or 5.0 mg/kg), in combination with 5 min of restraint, also reduced L/M ratios of hormonally primed, ovariectomized rats. The neural sites responsible for ketanserin's additivity with restraint are unknown, but infusion of the drug into the ventromedial nucleus of the hypothalamus (VMN) did not mimic the systemic treatment. However, 5 min of restraint did enhance the effects of VMN infusion with the 5-HT(1A) receptor agonist, 8-OH-DPAT. In contrast, 8-OH-DPAT's systemic potency was not enhanced by restraint. These findings support the hypothesis that a mild stressor increases the lordosis-inhibiting effects of 5-HT(1A) receptor agonists and that 5-HT(2) receptors may protect against such disruption of lordosis behavior.