Transcription-independent functions of p53 in DNA repair pathway selection.

Recently discovered transcription-independent features of p53 involve the choice of DNA damage repair pathway after PARylation, and p53's complex formation with phosphoinositide lipids, PI(4,5)P2 . PARylation-mediated rapid accumulation of p53 at DNA damage sites is linked to the recruitment of downstream repair factors and tumor suppression. This links p53's capability to sense damaged DNA in vitro and its relevant functions in cells. Further, PI(4,5)P2 rapidly accumulates at damage sites like p53 and complexes with p53, while it is required for ATR recruitment. These findings help explain how p53 and PI(4,5)P2 maintain genome stability by directing DNA repair pathway choice. Additionally, there is a strong correlation between p53 sequence homology, genome mutation rates as well as lifespans across various mammalian species. Further investigation is required to better understand the connections between genome stability, tumor suppression, longevity and the transcriptional-independent function of p53.

When PIP2 Meets p53: Nuclear Phosphoinositide Signaling in the DNA Damage Response.

The mechanisms that maintain genome stability are critical for preventing tumor progression. In the past decades, many strategies were developed for cancer treatment to disrupt the DNA repair machinery or alter repair pathway selection. Evidence indicates that alterations in nuclear phosphoinositide lipids occur rapidly in response to genotoxic stresses. This implies that nuclear phosphoinositides are an upstream element involved in DNA damage signaling. Phosphoinositides constitute a new signaling interface for DNA repair pathway selection and hence a new opportunity for developing cancer treatment strategies. However, our understanding of the underlying mechanisms by which nuclear phosphoinositides regulate DNA damage repair, and particularly the dynamics of those processes, is rather limited. This is partly because there are a limited number of techniques that can monitor changes in the location and/or abundance of nuclear phosphoinositide lipids in real time and in live cells. This review summarizes our current knowledge regarding the roles of nuclear phosphoinositides in DNA damage response with an emphasis on the dynamics of these processes. Based upon recent findings, there is a novel model for p53's role with nuclear phosphoinositides in DNA damage response that provides new targets for synthetic lethality of tumors.

Rapid recruitment of p53 to DNA damage sites directs DNA repair choice and integrity.

SignificanceOur work focuses on the critical longstanding question of the nontranscriptional role of p53 in tumor suppression. We demonstrate here that poly(ADP-ribose) polymerase (PARP)-dependent modification of p53 enables rapid recruitment of p53 to damage sites, where it in turn directs early repair pathway selection. Specifically, p53-mediated recruitment of 53BP1 at early time points promotes nonhomologous end joining over the more error-prone microhomology end-joining. Similarly, p53 directs nucleotide excision repair by mediating DDB1 recruitment. This property of p53 also correlates with tumor suppression in vivo. Our study provides mechanistic insight into how certain transcriptionally deficient p53 mutants may retain tumor-suppressive functions through regulating the DNA damage response.

Association between Flow-Mediated Dilation and Skin Perfusion Pressure with Peripheral Artery Disease in Hemodialysis Patients.

Flow-mediated dilation (FMD) is used to noninvasively assess the health of blood vessels and it has been shown to have a similar predictive ability for cardiovascular disease to traditional risk factors. Skin perfusion pressure (SPP) refers to the blood pressure required to restore capillary or microcirculatory flow after controlled occlusion and the return of flow. SPP has been shown to be an important measurement when making clinical decisions for patients with limb ischemia and to be a predictor of the likelihood of wound healing. Peripheral artery disease is common in hemodialysis (HD) patients. However, little is known about the association between FMD or SPP and peripheral artery disease. The aim of this study was to evaluate the association between FMD and SPP with brachial-ankle pulse wave velocity (baPWV) and ankle-brachial index (ABI) in HD patients in Taiwan, an area with a high rate of ESRD. This study was conducted at a regional hospital in southern Taiwan. ABI and baPWV values were measured using an ABI automated device. FMD and SPP were measured using ultrasound and a microvasculature blood flow monitor, respectively. Eighty patients were enrolled in this study. Compared to the patients with an ABI ≥ 0.95, those with an ABI < 0.95 had lower SPP of the feet (dorsal and plantar portions, both p < 0.001). After multivariable adjustments, low triglycerides (p = 0.033) and high calcium-phosphate product (p = 0.018) were significantly associated with low FMD. Further, low ABI (p = 0.001) and low baPWV (p = 0.036) were significantly associated with low SPP of dorsal portions. Old age (p = 0.005), low high-density lipoprotein cholesterol (p = 0.016), and low ABI (p = 0.002) were significantly associated with low SPP of plantar portions. This study demonstrated an association between FMD and SPP with peripheral artery disease in HD patients. Patients with low ABI and baPWV had a high risk of low SPP of the feet. However, there was no significant correlation between FMD and ABI or baPWV.

Selective killing of transformed cells by mechanical stretch.

Cancer cells differ from normal cells in several important features like anchorage independence, Warburg effect and mechanosensing. Further, in recent studies, they respond aberrantly to external mechanical distortion. Consistent with altered mechano-responsiveness, we find that cyclic stretching of tumor cells from many different tissues reduces growth rate and causes apoptosis on soft surfaces. Surprisingly, normal cells behave similarly when transformed by depletion of the rigidity sensor protein (Tropomyosin 2.1). Restoration of rigidity sensing in tumor cells promotes rigidity dependent mechanical behavior, i.e. cyclic stretching enhances growth and reduces apoptosis on soft surfaces. The mechanism of mechanical apoptosis (mechanoptosis) of transformed cells involves calcium influx through the mechanosensitive channel, Piezo1 that activates calpain 2 dependent apoptosis through the BAX molecule and subsequent mitochondrial activation of caspase 3 on both fibronetin and collagen matrices. Thus, it is possible to selectively kill tumor cells by mechanical perturbations, while stimulating the growth of normal cells.

Polyelectrolyte Gels Formed by Filamentous Biopolymers: Dependence of Crosslinking Efficiency on the Chemical Softness of Divalent Cations.

Filamentous anionic polyelectrolytes are common in biological materials. Some examples are the cytoskeletal filaments that assemble into networks and bundled structures to give the cell mechanical resistance and that act as surfaces on which enzymes and other molecules can dock. Some viruses, especially bacteriophages are also long thin polyelectrolytes, and their bending stiffness is similar to those of the intermediate filament class of cytoskeletal polymers. These relatively stiff, thin, and long polyelectrolytes have charge densities similar to those of more flexible polyelectrolytes such as DNA, hyaluronic acid, and polyacrylates, and they can form interpenetrating networks and viscoelastic gels at volume fractions far below those at which more flexible polymers form hydrogels. In this report, we examine how different types of divalent and multivalent counterions interact with two biochemically different but physically similar filamentous polyelectrolytes: Pf1 virus and vimentin intermediate filaments (VIF). Different divalent cations aggregate both polyelectrolytes similarly, but transition metal ions are more efficient than alkaline earth ions and their efficiency increases with increasing atomic weight. Comparison of these two different types of polyelectrolyte filaments enables identification of general effects of counterions with polyelectrolytes and can identify cases where the interaction of the counterions and the filaments exhibits stronger and more specific interactions than those of counterion condensation.

Reciprocal regulation of actomyosin organization and contractility in nonmuscle cells by tropomyosins and alpha-actinins.

Contractile arrays of actin and myosin II filaments drive many essential processes in nonmuscle cells, including migration and adhesion. Sequential organization of actin and myosin along one dimension is followed by expansion into a two-dimensional network of parallel actomyosin fibers, in which myosin filaments are aligned to form stacks. The process of stack formation has been studied in detail. However, factors that oppose myosin stack formation have not yet been described. Here, we show that tropomyosins act as negative regulators of myosin stack formation. Knockdown of any or all tropomyosin isoforms in rat embryonic fibroblasts resulted in longer and more numerous myosin stacks and a highly ordered actomyosin organization. The molecular basis for this, we found, is the competition between tropomyosin and alpha-actinin for binding actin. Surprisingly, excessive order in the actomyosin network resulted in smaller focal adhesions, lower tension within the network, and smaller traction forces. Conversely, disordered actomyosin bundles induced by alpha-actinin knockdown led to higher than normal tension and traction forces. Thus, tropomyosin acts as a check on alpha-actinin to achieve intermediate levels of myosin stacks matching the force requirements of the cell.

Lateral distribution of phosphatidylinositol 4,5-bisphosphate in membranes regulates formin- and ARP2/3-mediated actin nucleation.

Spatial and temporal control of actin polymerization is fundamental for many cellular processes, including cell migration, division, vesicle trafficking, and response to agonists. Many actin-regulatory proteins interact with phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) and are either activated or inactivated by local PI(4,5)P2 concentrations that form transiently at the cytoplasmic face of cell membranes. The molecular mechanisms of these interactions and how the dozens of PI(4,5)P2-sensitive actin-binding proteins are selectively recruited to membrane PI(4,5)P2 pools remains undefined. Using a combination of biochemical, imaging, and cell biologic studies, combined with molecular dynamics and analytical theory, we test the hypothesis that the lateral distribution of PI(4,5)P2 within lipid membranes and native plasma membranes alters the capacity of PI(4,5)P2 to nucleate actin assembly in brain and neutrophil extracts and show that activities of formins and the Arp2/3 complex respond to PI(4,5)P2 lateral distribution. Simulations and analytical theory show that cholesterol promotes the cooperative interaction of formins with multiple PI(4,5)P2 headgroups in the membrane to initiate actin nucleation. Masking PI(4,5)P2 with neomycin or disrupting PI(4,5)P2 domains in the plasma membrane by removing cholesterol decreases the ability of these membranes to nucleate actin assembly in cytoplasmic extracts.

DNA damage causes rapid accumulation of phosphoinositides for ATR signaling.

Phosphoinositide lipids (PPIs) are enriched in the nucleus and are accumulated at DNA damage sites. Here, we investigate roles of nuclear PPIs in DNA damage response by sequestering specific PPIs with the expression of nuclear-targeted PH domains, which inhibits recruitment of Ataxia telangiectasia and Rad3-related protein (ATR) and reduces activation of Chk1. PPI-binding domains rapidly (< 1 s) accumulate at damage sites with local enrichment of PPIs. Accumulation of PIP3 in complex with the nuclear receptor protein, SF1, at damage sites requires phosphorylation by inositol polyphosphate multikinase (IPMK) and promotes nuclear actin assembly that is required for ATR recruitment. Suppressed ATR recruitment/activation is confirmed with latrunculin A and wortmannin treatment as well as IPMK or SF1 depletion. Other DNA repair pathways involving ATM and DNA-PKcs are unaffected by PPI sequestration. Together, these findings reveal that nuclear PPI metabolism mediates an early damage response through the IPMK-dependent pathway to specifically recruit ATR.

Cholesterol-Dependent Phase-Demixing in Lipid Bilayers as a Switch for the Activity of the Phosphoinositide-Binding Cytoskeletal Protein Gelsolin.

The lateral distribution of phosphatidylinositol 4,5-bisphosphate (PIP2) in lipid bilayers is affected both by divalent cation-mediated attractions and cholesterol-dependent phase demixing. The effects of lateral redistribution of PIP2 within a membrane on PIP2-protein interactions are explored with an N-terminal fragment of gelsolin (NtGSN) that severs actin in a Ca(2+)-insensitive manner. The extent of NtGSN inhibition by PIP2-containing large unilamellar vesicles (LUVs) depends on the lateral organization of the membrane as quantified by an actin-severing assay. At a fixed PIP2 mole fraction, the inhibition is largely enhanced by the segregation of liquid ordered/liquid disordered (Lo/Ld) phases that is induced by altering either cholesterol content or temperature, whereas the presence of Ca(2+) only slightly improves the inhibition. Inhibition of gelsolin induced by demixed LUVs is more effective with decreasing temperature, coincident with increasing membrane order as determined by Laurdan generalized polarization and is reversible as the temperature increases. This result suggests that PIP2-mediated inhibition of gelsolin function depends not only on changes in global concentration but also on lateral distribution of PIP2. These observations imply that gelsolin, and perhaps other PIP2-regulated proteins, can be activated or inactivated by the formation of nanodomains or clusters without changing PIP2 bulk concentration in the cell membrane.

Integrin-beta3 clusters recruit clathrin-mediated endocytic machinery in the absence of traction force.

The turnover of integrin receptors is critical for cell migration and adhesion dynamics. Here we find that force development at integrins regulates adaptor protein recruitment and endocytosis. Using mobile RGD (Arg-Gly-Asp) ligands on supported lipid membranes (RGD membranes) and rigid RGD ligands on glass (RGD-glass), we find that matrix force-dependent integrin signals block endocytosis. Dab2, an adaptor protein of clathrin-mediated endocytosis, is not recruited to activated integrin-beta3 clusters on RGD-glass; however, it is recruited to integrin-mediated adhesions on RGD membranes. Further, when force generation is inhibited on RGD-glass, Dab2 binds to integrin-beta3 clusters. Dab2 binding to integrin-beta3 excludes other adhesion-related adaptor proteins, such as talin. The clathrin-mediated endocytic machinery combines with Dab2 to facilitate the endocytosis of RGD-integrin-beta3 clusters. From these observations, we propose that loss of traction force on ligand-bound integrin-beta3 causes recruitment of Dab2/clathrin, resulting in endocytosis of integrins.

Physical chemistry and membrane properties of two phosphatidylinositol bisphosphate isomers.

The most highly charged phospholipids, polyphosphoinositides, are often involved in signaling pathways that originate at cell-cell and cell-matrix contacts, and different isomers of polyphosphoinositides have distinct biological functions that cannot be explained by separate highly specific protein ligand binding sites [Lemmon, Nat. Rev. Mol. Cell Biol., 2008, 9, 99-111]. PtdIns(3,5)P2 is a low abundance phosphoinositide localized to cytoplasmic-facing membrane surfaces, with relatively few known ligands, yet PtdIns(3,5)P2 plays a key role in controlling membrane trafficking events and cellular stress responses that cannot be duplicated by other phosphoinositides [Dove et al., Nature, 1997, 390, 187-192; Michell, FEBS J., 2013, 280, 6281-6294]. Here we show that PtdIns(3,5)P2 is structurally distinct from PtdIns(4,5)P2 and other more common phospholipids, with unique physical chemistry. Using multiscale molecular dynamics techniques on the quantum level, single molecule, and in bilayer settings, we found that the negative charge of PtdIns(3,5)P2 is spread over a larger area, compared to PtdIns(4,5)P2, leading to a decreased ability to bind divalent ions. Additionally, our results match well with experimental data characterizing the cluster forming potential of these isomers in the presence of Ca(2+) [Wang et al., J. Am. Chem. Soc., 2012, 134, 3387-3395; van den Bogaart et al., Nature, 2011, 479, 552-555]. Our results demonstrate that the different cellular roles of PtdIns(4,5)P2 and PtdIns(3,5)P2in vivo are not simply determined by their localization by enzymes that produce or degrade them, but also by their molecular size, ability to chelate ions, and the partial dehydration of those ions, which might affect the ability of PtdIns(3,5)P2 and PtdIns(4,5)P2 to form phosphoinositide-rich clusters in vitro and in vivo.

Polyelectrolyte properties of filamentous biopolymers and their consequences in biological fluids.

Anionic polyelectrolyte filaments are common in biological cells. DNA, RNA, the cytoskeletal filaments F-actin, microtubules, and intermediate filaments, and polysaccharides such as hyaluronan that form the pericellular matrix all have large net negative charge densities distributed over their surfaces. Several filamentous viruses with diameters and stiffnesses similar to those of cytoskeletal polymers also have similar negative charge densities. Extracellular protein filaments such collagen, fibrin and elastin, in contrast, have notably smaller charge densities and do not behave as highly charged polyelectrolytes in solution. This review summarizes data that demonstrate generic counterion-mediated effects on four structurally unrelated biopolymers of similar charge density: F-actin, vimentin, Pf1 virus, and DNA, and explores the possible biological and pathophysiological consequences of the polyelectrolyte properties of biological filaments.

Structural basis for PI(4)P-specific membrane recruitment of the Legionella pneumophila effector DrrA/SidM.

Recruitment of the Legionella pneumophila effector DrrA to the Legionella-containing vacuole, where it activates and AMPylates Rab1, is mediated by a P4M domain that binds phosphatidylinositol 4-phosphate [PI(4)P] with high affinity and specificity. Despite the importance of PI(4)P in Golgi trafficking and its manipulation by pathogens, the structural bases for PI(4)P-dependent membrane recruitment remain poorly defined. Here, we determined the crystal structure of a DrrA fragment including the P4M domain in complex with dibutyl PI(4)P and investigated the determinants of phosphoinositide recognition and membrane targeting. Headgroup recognition involves an elaborate network of direct and water-mediated interactions with basic and polar residues in the context of a deep, constrictive binding pocket. An adjacent hydrophobic helical element packs against the acyl chains and inserts robustly into PI(4)P-containing monolayers. The structural, biochemical, and biophysical data reported here support a detailed structural mechanism for PI(4)P-dependent membrane targeting by DrrA.

Counterion-mediated pattern formation in membranes containing anionic lipids.

Most lipid components of cell membranes are either neutral, like cholesterol, or zwitterionic, like phosphatidylcholine and sphingomyelin. Very few lipids, such as sphingosine, are cationic at physiological pH. These generally interact only transiently with the lipid bilayer, and their synthetic analogs are often designed to destabilize the membrane for drug or DNA delivery. However, anionic lipids are common in both eukaryotic and prokaryotic cell membranes. The net charge per anionic phospholipid ranges from -1 for the most abundant anionic lipids such as phosphatidylserine, to near -7 for phosphatidylinositol 3,4,5 trisphosphate, although the effective charge depends on many environmental factors. Anionic phospholipids and other negatively charged lipids such as lipopolysaccharides are not randomly distributed in the lipid bilayer, but are highly restricted to specific leaflets of the bilayer and to regions near transmembrane proteins or other organized structures within the plane of the membrane. This review highlights some recent evidence that counterions, in the form of monovalent or divalent metal ions, polyamines, or cationic protein domains, have a large influence on the lateral distribution of anionic lipids within the membrane, and that lateral demixing of anionic lipids has effects on membrane curvature and protein function that are important for biological control.

Counterion-mediated cluster formation by polyphosphoinositides.

Polyphosphoinositides (PPI) and in particular PI(4,5)P2, are among the most highly charged molecules in cell membranes, are important in many cellular signaling pathways, and are frequently targeted by peripheral polybasic proteins for anchoring through electrostatic interactions. Such interactions between PIP2 and proteins containing polybasic stretches depend on the physical state and the lateral distribution of PIP2 within the inner leaflet of the cell's lipid bilayer. The physical and chemical properties of PIP2 such as pH-dependent changes in headgroup ionization and area per molecule as determined by experiments together with molecular simulations that predict headgroup conformations at various ionization states have revealed the electrostatic properties and phase behavior of PIP2-containing membranes. This review focuses on recent experimental and computational developments in defining the physical chemistry of PIP2 and its interactions with counterions. Ca(2+)-induced changes in PIP2 charge, conformation, and lateral structure within the membrane are documented by numerous experimental and computational studies. A simplified electrostatic model successfully predicts the Ca(2+)-driven formation of PIP2 clusters but cannot account for the different effects of Ca(2+) and Mg(2+) on PIP2-containing membranes. A more recent computational study is able to see the difference between Ca(2+) and Mg(2+) binding to PIP2 in the absence of a membrane and without cluster formation. Spectroscopic studies suggest that divalent cation- and multivalent polyamine-induced changes in the PIP2 lateral distribution in model membrane are also different, and not simply related to the net charge of the counterion. Among these differences is the capacity of Ca(2+) but not other polycations to induce nm scale clusters of PIP2 in fluid membranes. Recent super resolution optical studies show that PIP2 forms nanoclusters in the inner leaflet of a plasma membrane with a similar size distribution as those induced by Ca(2+) in model membranes. The mechanisms by which PIP2 forms nanoclusters and other structures inside a cell remain to be determined, but the unique electrostatic properties of PIP2 and its interactions with multivalent counterions might have particular physiological relevance.

Divalent cation-induced cluster formation by polyphosphoinositides in model membranes.

Polyphosphoinositides (PPIs) and in particular phosphatidylinositol-(4,5)-bisphosphate (PI4,5P2), control many cellular events and bind with variable levels of specificity to hundreds of intracellular proteins in vitro. The much more restricted targeting of proteins to PPIs in cell membranes is thought to result in part from the formation of spatially distinct PIP2 pools, but the mechanisms that cause formation and maintenance of PIP2 clusters are still under debate. The hypothesis that PIP2 forms submicrometer-sized clusters in the membrane by electrostatic interactions with intracellular divalent cations is tested here using lipid monolayer and bilayer model membranes. Competitive binding between Ca(2+) and Mg(2+) to PIP2 is quantified by surface pressure measurements and analyzed by a Langmuir competitive adsorption model. The physical chemical differences among three PIP2 isomers are also investigated. Addition of Ca(2+) but not Mg(2+), Zn(2+), or polyamines to PIP2-containing monolayers induces surface pressure drops coincident with the formation of PIP2 clusters visualized by fluorescence, atomic force, and electron microscopy. Studies of bilayer membranes using steady-state probe-partitioning fluorescence resonance energy transfer (SP-FRET) and fluorescence correlation spectroscopy (FCS) also reveal divalent metal ion (Me(2+))-induced cluster formation or diffusion retardation, which follows the trend: Ca(2+) ≫ Mg(2+) > Zn(2+), and polyamines have minimal effects. These results suggest that divalent metal ions have substantial effects on PIP2 lateral organization at physiological concentrations, and local fluxes in their cytoplasmic levels can contribute to regulating protein-PIP2 interactions.

Divalent cation-dependent formation of electrostatic PIP2 clusters in lipid monolayers.

Polyphosphoinositides are among the most highly charged molecules in the cell membrane, and the most common polyphosphoinositide, phosphatidylinositol-4,5-bisphosphate (PIP(2)), is involved in many mechanical and biochemical processes in the cell membrane. Divalent cations such as calcium can cause clustering of the polyanionic PIP(2), but the origin and strength of the effective attractions leading to clustering has been unclear. In addition, the question of whether the ion-mediated attractions could be strong enough to alter the mechanical properties of the membrane, to our knowledge, has not been addressed. We study phase separation in mixed monolayers of neutral and highly negatively charged lipids, induced by the addition of divalent positively charged counterions, both experimentally and numerically. We find good agreement between experiments on mixtures of PIP(2) and 1-stearoyl-2-oleoyl phosphatidylcholine and simulations of a simplified model in which only the essential electrostatic interactions are retained. In addition, we find numerically that under certain conditions the effective attractions can rigidify the resulting clusters. Our results support an interpretation of PIP(2) clustering as governed primarily by electrostatic interactions. At physiological pH, the simulations suggest that the effective attractions are strong enough to give nearly pure clusters of PIP(2) even at small overall concentrations of PIP(2).

Gelation of semiflexible polyelectrolytes by multivalent counterions.

Filamentous polyelectrolytes in aqueous solution aggregate into bundles by interactions with multivalent counterions. These effects are well documented by experiment and theory. Theories also predict a gel phase in isotropic rodlike polyelectrolyte solutions caused by multivalent counterion concentrations much lower than those required for filament bundling. We report here the gelation of Pf1 virus, a model semiflexible polyelectrolyte, by the counterions Mg(2+), Mn(2+) and spermine(4+). Gelation can occur at 0.04% Pf1 volume fraction, which is far below the isotropic-nematic transition of 0.7% for Pf1 in monovalent salt. Unlike strongly crosslinked gels of semiflexible polymers, which stiffen at large strains, Pf1 gels reversibly soften at high strain. The onset strain for softening depends on the strength of interaction between counterions and the polyelectrolyte. Simulations show that the elasticity of counterion crosslinked gels is consistent with a model of semiflexible filaments held by weak crosslinks that reversibly rupture at a critical force.

Kinase associated-1 domains drive MARK/PAR1 kinases to membrane targets by binding acidic phospholipids.

Phospholipid-binding modules such as PH, C1, and C2 domains play crucial roles in location-dependent regulation of many protein kinases. Here, we identify the KA1 domain (kinase associated-1 domain), found at the C terminus of yeast septin-associated kinases (Kcc4p, Gin4p, and Hsl1p) and human MARK/PAR1 kinases, as a membrane association domain that binds acidic phospholipids. Membrane localization of isolated KA1 domains depends on phosphatidylserine. Using X-ray crystallography, we identified a structurally conserved binding site for anionic phospholipids in KA1 domains from Kcc4p and MARK1. Mutating this site impairs membrane association of both KA1 domains and intact proteins and reveals the importance of phosphatidylserine for bud neck localization of yeast Kcc4p. Our data suggest that KA1 domains contribute to "coincidence detection," allowing kinases to bind other regulators (such as septins) only at the membrane surface. These findings have important implications for understanding MARK/PAR1 kinases, which are implicated in Alzheimer's disease, cancer, and autism.