Masha Prager-Khoutorsky

Fibroblast polarization is a matrix-rigidity-dependent process controlled by focal adhesion mechanosensing

Cell elongation and polarization are basic morphogenetic responses to extracellular matrix adhesion. We demonstrate here that human cultured fibroblasts readily polarize when plated on rigid, but not on compliant, substrates. On rigid surfaces, large and uniformly oriented focal adhesions are formed, whereas cells plated on compliant substrates form numerous small and radially oriented adhesions. Live-cell monitoring showed that focal adhesion alignment precedes the overall elongation of the cell, indicating that focal adhesion orientation may direct cell polarization. siRNA-mediated knockdown of 85 human protein tyrosine kinases (PTKs) induced distinct alterations in the cell polarization response, as well as diverse changes in cell traction force generation and focal adhesion formation. Remarkably, changes in rigidity-dependent traction force development, or focal adhesion mechanosensing, were consistently accompanied by abnormalities in the cell polarization response. We propose that the different stages of cell polarization are regulated by multiple, PTK-dependent molecular checkpoints that jointly control cell contractility and focal-adhesion-mediated mechanosensing.

Pharmacogenetic Inhibition of eIF4E-Dependent Mmp9 mRNA Translation Reverses Fragile X Syndrome-like Phenotypes

SUMMARY Fragile X syndrome (FXS) is the leading genetic cause of autism. Mutations in Fmr1 (fragile X mental retardation 1 gene) engender exaggerated translation resulting in dendritic spine dysmorphogenesis, synaptic plasticity alterations, and behavioral deficits in mice, which are reminiscent of FXS pheno-types. Using postmortem brains from FXS patients and Fmr1 knockout mice (Fmr1 −/y), we show that phosphorylation of the mRNA 5′ cap binding protein, eukaryotic initiation factor 4E (eIF4E), is elevated concomitant with increased expression of matrix metalloproteinase 9 (MMP-9) protein. Genetic or pharmacological reduction of eIF4E phosphorylation rescued core behavioral deficits, synaptic plasticity alterations, and dendritic spine morphology defects via reducing exaggerated translation of Mmp9 mRNA in Fmr1 −/y mice, whereas MMP-9 overexpression produced several FXS-like phenotypes. These results uncover a mechanism of regulation of synaptic function by translational control of Mmp-9 in FXS, which opens the possibility of new treatment avenues for the diverse neurological and psychiatric aspects of FXS.

Control of Synaptic Plasticity and Memory via Suppression of Poly(A)-Binding Protein

Control of protein synthesis is critical for synaptic plasticity and memory formation. However, the molecular mechanisms linking neuronal activity to activation of mRNA translation are not fully understood. Here, we report that the translational repressor poly(A)-binding protein (PABP)-interacting protein 2A (PAIP2A), an inhibitor of PABP, is rapidly proteolyzed by calpains in stimulated neurons and following training for contextual memory. Paip2a knockout mice exhibit a lowered threshold for the induction of sustained long-term potentiation and an enhancement of long-term memory after weak training. Translation of CaMKIIα mRNA is enhanced in Paip2a⁻/⁻ slices upon tetanic stimulation and in the hippocampus of Paip2a⁻/⁻ mice following contextual fear learning. We demonstrate that activity-dependent degradation of PAIP2A relieves translational inhibition of memory-related genes through PABP reactivation and conclude that PAIP2A is a pivotal translational regulator of synaptic plasticity and memory.

Unique Interweaved Microtubule Scaffold Mediates Osmosensory Transduction via Physical Interaction with TRPV1

The electrical activity of mammalian osmosensory neurons (ONs) is increased by plasma hypertonicity to command thirst, antidiuretic hormone release, and increased sympathetic tone during dehydration. Osmosensory transduction is a mechanical process whereby decreases in cell volume cause the activation of transient receptor potential vanilloid type-1 (TRPV1) channels to induce depolarization and increase spiking activity in ONs. However, it is not known how cell shrinking is mechanically coupled to channel activation. Using superresolution imaging, we found that ONs are endowed with a uniquely interweaved scaffold of microtubules throughout their somata. Microtubules physically interact with the C terminus of TRPV1 at the cell surface and provide a pushing force that drives channels activation during shrinking. Moreover, we found that changes in the density of these interactions can bidirectionally modulate osmosensory gain. Microtubules are thus an essential component of the vital neuronal mechanotransduction apparatus that allows the brain to monitor and correct body hydration.

ΔN-TRPV1: A Molecular Co-detector of Body Temperature and Osmotic Stress

Thirst and antidiuretic hormone secretion occur during hyperthermia or hypertonicity to preserve body hydration. These vital responses are triggered when hypothalamic osmoregulatory neurons become depolarized by ion channels encoded by an unknown product of the transient receptor potential vanilloid-1 gene (Trpv1). Here, we show that rodent osmoregulatory neurons express a transcript of Trpv1 that mediates the selective translation of a TRPV1 variant that lacks a significant portion of the channels amino terminus (ΔN-TRPV1). The mRNA transcript encoding this variant (Trpv1dn) is widely expressed in the brains of osmoregulating vertebrates, including the human hypothalamus. Transfection of Trpv1dn into heterologous cells induced the expression of ion channels that could be activated by either hypertonicity or by heating in the physiological range. Moreover, expression of Trpv1dn rescued the osmosensory and thermosensory responses of single hypothalamic neurons obtained from Trpv1 knockout mice. ΔN-TRPV1 is therefore a co-detector of core body temperature and fluid tonicity.

Osmosensation in vasopressin neurons: changing actin density to optimize function

The proportional relation between circulating vasopressin concentration and plasma osmolality is fundamental for body fluid homeostasis. Although changes in the sensitivity of this relation are associated with pathophysiological conditions, central mechanisms modulating osmoregulatory gain are unknown. Here, we review recent data that sheds important light on this process. The cell autonomous osmosensitivity of vasopressin neurons depends on cation channels comprising a variant of the transient receptor potential vanilloid 1 (TRPV1) channel. Hyperosmotic activation is mediated by a mechanical process where sensitivity increases in proportion with actin filament density. Moreover, angiotensin II amplifies osmotic activation by a rapid stimulation of actin polymerization, suggesting that neurotransmitter-induced changes in cytoskeletal organization in osmosensory neurons can mediate central changes in osmoregulatory gain.

Multifaceted Regulation of Somatic Cell Reprogramming by mRNA Translational Control

Translational control plays a pivotal role in the regulation of the pluripotency network in embryonic stem cells, but its effect on reprogramming somatic cells to pluripotency has not been explored. Here, we show that eukaryotic translation initiation factor 4E (eIF4E) binding proteins (4E-BPs), which are translational repressors, have a multifaceted effect on the reprogramming of mouse embryonic fibroblasts (MEFs) into induced pluripotent stem cells (iPSCs). Loss of 4E-BP expression attenuates the induction of iPSCs at least in part through increased translation of p21, a known inhibitor of somatic cell reprogramming. However, MEFs lacking both p53 and 4E-BPs show greatly enhanced reprogramming resulting from a combination of reduced p21 transcription and enhanced translation of endogenous mRNAs such as Sox2 and Myc and can be reprogrammed through the expression of only exogenous Oct4. Thus, 4E-BPs exert both positive and negative effects on reprogramming, highlighting the key role that translational control plays in regulating this process.

Signaling function of α-catenin in microtubule regulation

Centrosomes control microtubule dynamics in many cell types, and their removal from the cytoplasm leads to a shift from dynamic instability to treadmilling behavior and to a dramatic decrease of microtubule mass (Rodionov et al.1999, PNAS 96:115). In cadherin-expressing cells, these effects can be reversed: non-centrosomal cytoplasts that form cadherin-mediated adherens junctions display dense arrays of microtubules (Chausovsky et al. 2000, Nature Cell Biol. 2:797). In adherens junctions, cadherins cytoplasmic domain binds p120 catenin and β-catenin, which in turn binds α-catenin. To elucidate the roles of the cadherin-associated proteins in regulating microtubule dynamics, we prepared GFP-tagged, plasma membrane targeted or untargeted p120 catenin, α-catenin, and β-catenin and tested their ability to rescue the loss of microtubule mass caused by centrosomal removal in the poorly adhesive cell line CHO-K1. Only membrane targeting of α-catenin showed a significant increase in microtubule length and density in centrosome-free cytoplasts. Expression of non-membrane-targeted α-catenin produced only a slight effect, while both membrane-targeted and non-targeted p120 and β-catenin were ineffective in this assay. Together, these findings suggest that α-catenin is able to regulate microtubule dynamics in a centrosome-independent manner.

eIF2α phosphorylation controls thermal nociception

Significance Distinct cellular stresses converge on the translation initiation factor, eukaryotic initiation factor 2α (eIF2α) to modulate the rate of protein synthesis. Increased phosphorylation of eIF2α has been described in peripheral neurons from neuropathic and diabetic rats. However, the role of eIF2α phosphorylation in pain has not been reported. Here we show that phosphorylation of eIF2α controls thermal, but not mechanical, sensation via modulation of the activity of a major heat transducer, transient receptor potential vanilloid 1. We also find that chronic inflammation-induced eIF2α phopshorylation contributes to inflammation-induced thermal hypersensitivity. These results demonstrate that eIF2α phosphorylation plays a major role in controlling noxious heat sensitivity. A response to environmental stress is critical to alleviate cellular injury and maintain cellular homeostasis. Eukaryotic initiation factor 2 (eIF2) is a key integrator of cellular stress responses and an important regulator of mRNA translation. Diverse stress signals lead to the phosphorylation of the α subunit of eIF2 (Ser51), resulting in inhibition of global protein synthesis while promoting expression of proteins that mediate cell adaptation to stress. Here we report that eIF2α is instrumental in the control of noxious heat sensation. Mice with decreased eIF2α phosphorylation (eIF2α+/S51A) exhibit reduced responses to noxious heat. Pharmacological attenuation of eIF2α phosphorylation decreases thermal, but not mechanical, pain sensitivity, whereas increasing eIF2α phosphorylation has the opposite effect on thermal nociception. The impact of eIF2α phosphorylation (p-eIF2α) on thermal thresholds is dependent on the transient receptor potential vanilloid 1. Moreover, we show that induction of eIF2α phosphorylation in primary sensory neurons in a chronic inflammation pain model contributes to thermal hypersensitivity. Our results demonstrate that the cellular stress response pathway, mediated via p-eIF2α, represents a mechanism that could be used to alleviate pathological heat sensation.

Effects of Peritoneal Sepsis on Rat Central Osmoregulatory Neurons Mediating Thirst and Vasopressin Release

Sepsis is a life-threatening condition caused by the systemic inflammatory response to a bacterial infection. Although much is known about the cellular and molecular changes that characterize the peripheral inflammatory response to sepsis, almost nothing is known of the neuronal changes that cause associated perturbations in the central control of homeostasis. Osmoregulation is one of the key homeostatic systems perturbed during sepsis. In healthy subjects, systemic hypertonicity normally excites osmoreceptor neurons in the organum vasculosum laminae terminalis (OVLT), which then activates downstream neurons that induce a parallel increase in water intake and arginine vasopressin (AVP) secretion to promote fluid expansion and maintain blood pressure. However, recent studies have shown that the early phase of sepsis is associated with increased AVP levels and suppressed thirst. Here we examined the electrophysiological properties of OVLT neurons and magnocellular neurosecretory cells (MNCs) in acute in vitro preparations obtained from rats subjected to sham surgery or cecal ligation and puncture (CLP). We found that the intrinsic excitability of OVLT neurons was not affected significantly 18–24 h after CLP. However, OVLT neurons in CLP rats were hyperpolarized significantly compared with shams. Moreover, a reduced proportion of these cells displayed spontaneous electrical activity and osmoresponsiveness in septic animals. In contrast, the osmoresponsiveness of MNCs was only attenuated by CLP, and a larger proportion of these neurons displayed spontaneous electrical activity in septic animals. These results suggest that acute sepsis disrupts centrally mediated osmoregulatory reflexes through differential effects on the properties of neurons in the OVLT and supraoptic nucleus. SIGNIFICANCE STATEMENT Sepsis is a life-threatening condition caused by the systemic inflammatory response to bacterial infection. Although the early phase of sepsis features impaired thirst and enhanced vasopressin release, the basis for these defects is unknown. Here, we show that cecal ligation and puncture (CLP) in rats impairs the osmoresponsiveness of neurons in the organum vasculosum lamina terminalis (OVLT; which drives thirst) and attenuates that of neurosecretory neurons in the supraoptic nucleus (SON; which secrete oxytocin and vasopressin). Notably, we found that OVLT neurons are hyperpolarized and electrically silenced. In contrast, CLP increased the proportion of SON neurons displaying spontaneous electrical activity. Therefore, CLP affects the properties of osmoregulatory neurons in a manner that can affect systemic osmoregulation.