Endocytic traffic in polarized epithelial cells: role of the actin and microtubule cytoskeleton

The cytoskeleton is required for multiple cellular events including endocytosis and the transfer of cargo within the endocytic system. Polarized epithelial cells are capable of endocytosis at either of their distinct apical or basolateral plasma membrane domains. Actin plays a role in internalization at both cell surfaces. Microtubules and actin are required for efficient transcytosis and delivery of proteins to late endosomes and lysosomes. Microtubules are also important in apical recycling pathways and, in some polarized cell types, basolateral recycling requires actin. The microtubule motor proteins dynein and kinesin and the class I unconventional myosin motors play a role in many of these trafficking steps. This review examines the endocytic pathways of polarized epithelial cells and focuses on the emerging roles of the actin cytoskeleton in these processes.     

Mid1ip1b modulates apical reorientation of non-centrosomal microtubule organizing center in epithelial cells

In most kinds of animal cells, the centrosome serves as the main microtubule organizing center (MTOC) that nucleates microtubule arrays throughout the cytoplasm to maintain cell structure, cell division and intracellular transport. Whereas in epithelial cells, non-centrosomal MTOCs are established in the apical domain for generating asymmetric microtubule fibers and cilia in epithelial cells for the organ morphogenesis during embryonic development. However, the mechanism by which MTOCs localize to the apical domain in epithelial cells remains largely unknown. Here, we show that Mid1ip1b has a close interaction with γ-tubulin protein, the central component of MTOC, and modulates lumen opening of the neural tube, gut, intestine, and kidney of zebrafish. Knockdown or dominant negative effect of Mid1ip1b resulted in failure of lumen formation of the organs as aforementioned. Moreover, the non-centrosomal MTOCs were unable to orientate to the apical domain in Mid1ip1b knockdown epithelial cells, and the centrosomal MTOCs were inaccurately placed in the apical domain, resulting in defective formation of asymmetric microtubules and misplacement of cilia in the apical domain. These data uncover a molecule that controls the proper localization of MTOCs in the apical domain in epithelial cells for organ morphogenesis during embryonic development.     

Breaking the epithelial polarity barrier in cancer: the strange case of LKB1/PAR-4

The PAR clan of polarity regulating genes was initially discovered in a genetic screen searching for genes involved in asymmetric cell divisions in the Caenorhabditis elegans embryo. Today, investigations in worms, flies and mammals have established PAR proteins as conserved and fundamental regulators of animal cell polarization in a broad range of biological phenomena requiring cellular asymmetries. The human homologue of invertebrate PAR-4, a serine–threonine kinase LKB1/STK11, has caught attention as a gene behind Peutz–Jeghers polyposis syndrome and as a bona fide tumour suppressor gene commonly mutated in sporadic cancer. LKB1 functions as a master regulator of AMP-activated protein kinase (AMPK) and 12 other kinases referred to as the AMPK-related kinases, including four human homologues of PAR-1. The role of LKB1 as part of the energy sensing LKB1-AMPK module has been intensively studied, whereas the polarity function of LKB1, in the context of homoeostasis or cancer, has gained less attention. Here, we focus on the PAR-4 identity of LKB1, discussing the weight of evidence indicating a role for LKB1 in regulation of cell polarity and epithelial integrity across species and highlight recent investigations providing new insight into the old question: does the PAR-4 identity of LKB1 matter in cancer?     

The role of PAR-1 in regulating the polarised microtubule cytoskeleton in the Drosophila follicular epithelium

The PAR-1 kinase plays a conserved role in cell polarity in C. elegans, Drosophila and mammals. We have investigated the role of PAR-1 in epithelial polarity by generating null mutant clones in the Drosophila follicular epithelium. Large clones show defects in apicobasal membrane polarity, but small clones induced later in development usually have a normal membrane polarity. However, all cells that lack PAR-1 accumulate spectrin and F-actin laterally, and show a strong increase in the density of microtubules. This is consistent with the observation that the mammalian PAR-1 homologues, the MARKs, dramatically reduce the number of microtubules, when overexpressed in tissue culture cells. The MARKs have been proposed to destabilize microtubules by inhibiting the stabilizing activity of the Tau family of microtubule-associated proteins. This is not the case in Drosophila, however, as null mutations in the single tau family member in the genome have no effect on the microtubule organisation in the follicle cells. Furthermore, PAR-1 activity stabilises microtubules, as microtubules in mutant cells depolymerise much more rapidly after cold or colcemid treatments. Loss of PAR-1 also disrupts the basal localisation of the microtubule plus ends, which are mislocalised to the centre of mutant cells. Thus, Drosophila PAR-1 regulates the density, stability and apicobasal organisation of microtubules. Although the direct targets of PAR-1 are unknown, we suggest that it functions by regulating the plus ends, possibly by capping them at the basal cortex.     

Solution structure of the kinase-associated domain 1 of mouse microtubule-associated protein/microtubule affinity-regulating kinase 3

Microtubule-associated protein/microtubule affinity-regulating kinases (MARKs)/PAR-1 are common regulators of cell polarity that are conserved from nematode to human. All of these kinases have a highly conserved C-terminal domain, which is termed the kinase-associated domain 1 (KA1), although its function is unknown. In this study, we determined the solution structure of the KA1 domain of mouse MARK3 by NMR spectroscopy. We found that approximately 50 additional residues preceding the previously defined KA1 domain are required for its proper folding. The newly defined KA1 domain adopts a compact alpha+beta structure with a betaalphabetabetabetabetaalpha topology. We also found a characteristic hydrophobic, concave surface surrounded by positively charged residues. This concave surface includes the highly conserved Glu-Leu-Lys-Leu motif at the C terminus, indicating that it is important for the function of the KA1 domain.     

Tight junction and polarity interaction in the transporting epithelial phenotype

Development of tight junctions and cell polarity in epithelial cells requires a complex cellular machinery to execute an internal program in response to ambient cues. Tight junctions, a product of this machinery, can act as gates of the paracellular pathway, fences that keep the identity of plasma membrane domains, bridges that communicate neighboring cells. The polarization internal program and machinery are conserved in yeast, worms, flies and mammals, and in cell types as different as epithelia, neurons and lymphocytes. Polarization and tight junctions are dynamic features that change during development, in response to physiological and pharmacological challenges and in pathological situations like infection.     

aPKC acts upstream of PAR-1b in both the establishment and maintenance of mammalian epithelial polarity

BACKGROUND: aPKC and PAR-1 are required for cell polarity in various contexts. In mammalian epithelial cells, aPKC localizes at tight junctions (TJs) and plays an indispensable role in the development of asymmetric intercellular junctions essential for the establishment and maintenance of apicobasal polarity. On the other hand, one of the mammalian PAR-1 kinases, PAR-1b/EMK1/MARK2, localizes to the lateral membrane in a complimentary manner with aPKC, but little is known about its role in apicobasal polarity of epithelial cells as well as its functional relationship with aPKC. RESULTS: We demonstrate that PAR-1b is essential for the asymmetric development of membrane domains of polarized MDCK cells. Nonetheless, it is not required for the junctional localization of aPKC nor the formation of TJs, suggesting that PAR-1b works downstream of aPKC during epithelial cell polarization. On the other hand, aPKC phosphorylates threonine 595 of PAR-1b and enhances its binding with 14-3-3/PAR-5. In polarized MDCK cells, T595 phosphorylation and 14-3-3 binding are observed only in the soluble form of PAR-1b, and okadaic acid treatment induces T595-dependent dissociation of PAR-1b from the lateral membrane. Furthermore, T595A mutation induces not only PAR-1b leakage into the apical membrane, but also abnormal development of membrane domains. These results suggest that in polarized epithelial cells, aPKC phosphorylates PAR-1b at TJs, and in cooperation with 14-3-3, promotes the dissociation of PAR-1b from the lateral membrane to regulate PAR-1b activity for the membrane domain development. CONCLUSIONS: These results suggest that mammalian aPKC functions upstream of PAR-1b in both the establishment and maintenance of epithelial cell polarity.     

The effects of diazepam on mitosis and the microtubule cytoskeleton II. Observations on newt epithelial and PtK1 cells

Summary The effects of diazepam (DZP) on mitosis and the microtubule (MT) cytoskeleton were examined using live and fixed PtK₁ and newt (Taricha granulosa) epithelial lung cells. DZP treatment caused rapid shortening of spindle MTs at prometaphase and metaphase, inducing movement of the poles together while chromosome oscillations continued. DZP treatment slowed the rate of anaphase A but did not detectably affect anaphase B, cell cleavage or interphase cells. Our results suggest that DZP inhibits mitosis by affecting prometaphase and metaphase MTs. Its action is not equivalent to that of common anti-MT drugs, since only a small subpopulation of MTs are significantly susceptible. Likewise, its effects are not equivalent to those generated by metabolic inhibitors. The related benzodiazepines, medazepam and oxazepam, induce effects equivalent to those of DZP.     

Dynamic regulation of GEF-H1 localization at microtubules by Par1b/MARK2

Par1b/MARK2 is a serine/threonine kinase that plays key roles in the development of cell polarity, but its precise mechanism of action remains unknown. Here we report that GEF-H1, a guanine nucleotide exchange factor for Rho-family small GTPases, is a novel substrate for Par1b. GEF-H1 directly associates with microtubules via its N-terminal C1 domain, which is known to regulate the activity of GEF-H1. Ectopically expressed GEF-H1 markedly promotes stabilization of microtubules, resulting in acetylation of microtubules. We find that Par1b phosphorylates GEF-H1 at three serine residues conserved in vertebrates and releases GEF-H1 from microtubules, which abrogates stabilization and acetylation of microtubules induced by GEF-H1 overexpression. The alanine mutant for the three phosphorylation sites (3SA) of GEF-H1 strongly induces stabilization and acetylation of microtubules, which was resistant to Par1b. Time-lapse imaging analyses reveal that GFP-fused GEF-H1 dynamically moved on microtubules from one protrusion to another, whereas the 3SA mutant was static. These data suggest that Par1b-phosphorylation regulates turnover of GEF-H1 localization by regulating its interaction with microtubules, which may contribute to cell polarization.     

Coordination of epithelial branching and salivary gland lumen formation by Wnt and FGF signals

Branching morphogenesis is a molecularly conserved mechanism that is adopted by several organs, such as the lung, kidney, mammary gland and salivary gland, to maximize the surface area of a tissue within a small volume. Branching occurs through repetitive clefting and elongation of spherical epithelial structures, called endbuds, which invade the surrounding mesenchyme. In the salivary gland, lumen formation takes place alongside branching morphogenesis, but in a controlled manner, so that branching is active at the distal ends of epithelial branches while lumen formation initiates at the proximal ends, and spreads distally. We present here data showing that interaction between FGF signaling and the canonical (β-catenin dependent) and non-canonical branches of Wnt signaling coordinates these two processes. Using the Axin2^lacZ reporter mice, we find Wnt/β-catenin signaling activity first in the mesenchyme and later, at the time of lumen formation, in the ductal epithelium. Gain and loss of function experiments reveal that this pathway exerts an inhibitory effect on salivary gland branching morphogenesis. We have found that endbuds remain devoid of Wnt/β-catenin signaling activity, a hallmark of ductal structures, through FGF-mediated inhibition of this pathway. Our data also show that FGF signaling has a major role in the control of lumen formation by preventing premature hollowing of epithelial endbuds and slowing down the canalization of presumptive ducts. Concomitantly, FGF signaling strongly represses the ductal marker Cp2l1, most likely via repression of Wnt5b and non-canonical Wnt signaling. Inhibition of canonical and non-canonical Wnt signaling in endbuds by FGF signaling occurs at least in part through sFRP1, a secreted inhibitor of Wnt signaling and downstream target of FGF signaling. Altogether, these findings point to a key function of FGF signaling in the maintenance of an undifferentiated state in endbud cells by inhibition of a ductal fate.     

DAPK activates MARK1/2 to regulate microtubule assembly, neuronal differentiation, and tau toxicity

Death-associated protein kinase (DAPK) is a key player in several modes of neuronal death/injury and has been implicated in the late-onset Alzheimer's disease (AD). DAPK promotes cell death partly through its effect on regulating actin cytoskeletons. In this study, we report that DAPK inhibits microtubule (MT) assembly by activating MARK/PAR-1 family kinases MARK1/2, which destabilize MT by phosphorylating tau and related MAP2/4. DAPK death domain, but not catalytic activity, is responsible for this activation by binding to MARK1/2 spacer region, thereby disrupting an intramolecular interaction that inhibits MARK1/2. Accordingly, DAPK(-/-) mice brain displays a reduction of tau phosphorylation and DAPK enhances the effect of MARK2 on regulating polarized neurite outgrowth. Using a well-characterized Drosophila model of tauopathy, we show that DAPK exerts an effect in part through MARK Drosophila ortholog PAR-1 to induce rough eye and loss of photoreceptor neurons. Furthermore, DAPK enhances tau toxicity through a PAR-1 phosphorylation-dependent mechanism. Together, our study reveals a novel mechanism of MARK activation, uncovers DAPK functions in modulating MT assembly and neuronal differentiation, and provides a molecular link of DAPK to tau phosphorylation, an event associated with AD pathology.     

Mirror‐symmetric microtubule assembly and cell interactions drive lumen formation in the zebrafish neural rod

By analysing the cellular and subcellular events that occur in the centre of the developing zebrafish neural rod, we have uncovered a novel mechanism of cell polarisation during lumen formation. Cells from each side of the neural rod interdigitate across the tissue midline. This is necessary for localisation of apical junctional proteins to the region where cells intersect the tissue midline. Cells assemble a mirror‐symmetric microtubule cytoskeleton around the tissue midline, which is necessary for the trafficking of proteins required for normal lumen formation, such as partitioning defective 3 and Rab11a to this point. This occurs in advance and is independent of the midline cell division that has been shown to have a powerful role in lumen organisation. To our knowledge, this is the first example of the initiation of apical polarisation part way along the length of a cell, rather than at a cell extremity. Although the midline division is not necessary for apical polarisation, it confers a morphogenetic advantage by efficiently eliminating cellular processes that would otherwise bridge the developing lumen.     

AMP-activated protein kinase induces actin cytoskeleton reorganization in epithelial cells

AMP-activated protein kinase (AMPK), a known regulator of cellular and systemic energy balance, is now recognized to control cell division, cell polarity and cell migration, all of which depend on the actin cytoskeleton. Here we report the effects of A769662, a pharmacological activator of AMPK, on cytoskeletal organization and signalling in epithelial Madin-Darby canine kidney (MDCK) cells. We show that AMPK activation induced shortening or radiation of stress fibers, uncoupling from paxillin and predominance of cortical F-actin. In parallel, Rho-kinase downstream targets, namely myosin regulatory light chain and cofilin, were phosphorylated. These effects resembled the morphological changes in MDCK cells exposed to hyperosmotic shock, which led to Ca²⁺-dependent AMPK activation via calmodulin-dependent protein kinase kinase-β(CaMKKβ), a known upstream kinase of AMPK. Indeed, hypertonicity-induced AMPK activation was markedly reduced by the STO-609 CaMKKβ inhibitor, as was the increase in MLC and cofilin phosphorylation. We suggest that AMPK links osmotic stress to the reorganization of the actin cytoskeleton.     

The apical localization of SGLT1 glucose transporter is determined by the short amino acid sequence in its N-terminal domain

SGLT1, an isoform of Na+-dependent glucose cotransporters, is localized at the apical plasma membrane in the epithelial cells of the small intestine and the kidney, where it plays a pivotal role in the absorption and reabsorption of sugars, respectively. To search the domain responsible for the apical localization of SGLT1, we constructed an N-terminal deletion clone series of rat SGLT1 and analyzed the localization of the respective products in Madin-Darby canine kidney (MDCK) cells. The products of N-terminal deletion clones up to the 19th amino acid were localized at the apical plasma membrane, whereas the products of N-terminal 20- and 23-amino-acid deletion clones were localized along the entire plasma membrane. Since single-amino-acid mutations of either D28N or D28G in the N-terminal domain give rise to glucose/galactose malabsorption disease, we examined the localization of these mutants. The products of D28N and D28G clones were localized in the cytoplasm, showing that the aspartic acid-28 may be essential for the delivery of SGLT1 to the plasma membrane. These results suggest that a short amino acid sequence of the N-terminal domain of SGLT1 plays important roles in plasma membrane targeting and specific apical localization of the protein.     

转谷氨酰胺酶2抑制H1亚型流感病毒在MDCK细胞中的增殖

组织转谷酰胺酶(transglutaminase 2,TGM2)是一种普遍存在的多功能蛋白,与不同细胞的粘附和肿瘤形成有关.有证据表明,TGM2参与了宿主细胞与病毒间的相互作用,但是对于流感病毒在细胞内增殖的影响还未有报道.为了探究MDCK细胞中TGM2对H1N1亚型流感病毒增殖的影响,本研究构建了TGM2过表达和敲除...     

Large gradient high magnetic field affects the association of MACF1 with actin and microtubule cytoskeleton

The intense inhomogeneous magnetic fields acting on the diamagnetic materials naturally present in cells can generate strong magnetic forces. We have developed a superconducting magnet platform with large gradient high magnetic field (LG‐HMF), which can produce three magnetic force fields of ?1360, 0, and 1312 T²/m, and three corresponding apparent gravity levels, namely 0, 1, and 2‐g for diamagnetic materials. In this study, the effects of different magnetic force fields on osteoblast‐like cells (MG‐63 and MC3T3‐E1) viability, microtubule actin crosslinking factor 1 (MACF1) expression and its association with cytoskeleton were investigated. Results showed that cell viability increased to different degrees after exposure to 0 or 1‐g conditions for 24 h, but it decreased by about 30% under 2‐g conditions compared with control conditions. An increase in MACF1 expression at the RNA or protein level was observed in osteoblast‐like cells under the magnetic force field of ?1360 T²/m (0‐g) relative to 1312 T²/m (2‐g). Under control conditions, anti‐MACF1 staining was scattered in the cytoplasm and partially colocalized with actin filaments (AFs) or microtubules (MTs) in the majority of osteoblast‐like cells. Under 0‐g conditions, MACF1 labeling was concentrated at perinuclear region and colocalization was not apparent. The patterns of anti‐MACF1 labeling on MTs varied with MTs' changing under LG‐HMF environment. In conclusion, LG‐HMF affects osteoblast‐like cell viability, MACF1 distribution, expression, and its association with cytoskeleton to some extent. Bioelectromagnetics 30:545–555, 2009. © 2009 Wiley‐Liss, Inc.     

Targeting of calcium:calmodulin signals to the cytoskeleton by IQGAP1

Mast cells reorganize their actin cytoskeleton in response to cytosolic calcium signals while in parallel secreting histamine and other inflammatory mediators. The effect of calcium on actin is mediated in large part through calmodulin. EGFP-tagged calmodulin is concentrated in the actin-rich cortex of RBL-2H3 mast cells. Transfection with small interfering RNA directed against the actin and calmodulin-binding protein IQGAP1 dramatically reduced expression of the latter protein and reduced or eliminated the concentration of calmodulin at the actin-rich cortex. Both actin reorganization and secretion were enhanced in IQGAP1 knockdown cells. Our results suggest a model in which calmodulin is targeted to and sequestered at the actin cytoskeleton by IQGAP1. Upon cell stimulation and the subsequent [Ca2+]i increase, it is immediately available to activate local downstream targets.     

A microtubule‐LUZP1 association around tight junction promotes epithelial cell apical constriction

Apical constriction is critical for epithelial morphogenesis, including neural tube formation. Vertebrate apical constriction is induced by di‐phosphorylated myosin light chain (ppMLC)‐driven contraction of actomyosin‐based circumferential rings (CRs), also known as perijunctional actomyosin rings, around apical junctional complexes (AJCs), mainly consisting of tight junctions (TJs) and adherens junctions (AJs). Here, we revealed a ppMLC‐triggered system at TJ‐associated CRs for vertebrate apical constriction involving microtubules, LUZP1, and myosin phosphatase. We first identified LUZP1 via unbiased screening of microtubule‐associated proteins in the AJC‐enriched fraction. In cultured epithelial cells, LUZP1 was found localized at TJ‐, but not at AJ‐, associated CRs, and LUZP1 knockout resulted in apical constriction defects with a significant reduction in ppMLC levels within CRs. A series of assays revealed that ppMLC promotes the recruitment of LUZP1 to TJ‐associated CRs, where LUZP1 spatiotemporally inhibits myosin phosphatase in a microtubule‐facilitated manner. Our results uncovered a hitherto unknown microtubule‐LUZP1 association at TJ‐associated CRs that inhibits myosin phosphatase, contributing significantly to the understanding of vertebrate apical constriction.     

ArfGAP1 restricts Mycobacterium tuberculosis entry by controlling the actin cytoskeleton

The interaction of Mycobacterium tuberculosis (Mtb) with pulmonary epithelial cells is critical for early stages of bacillus colonization and during the progression of tuberculosis. Entry of Mtb into epithelial cells has been shown to depend on F‐actin polymerization, though the molecular mechanisms are still unclear. Here, we demonstrate that mycobacterial uptake into epithelial cells requires rearrangements of the actin cytoskeleton, which are regulated by ADP‐ribosylation factor 1 (Arf1) and phospholipase D1 (PLD1), and is dependent on the M3 muscarinic receptor (M₃R). We show that this pathway is controlled by Arf GTPase‐activating protein 1 (ArfGAP1), as its silencing has an impact on actin cytoskeleton reorganization leading to uncontrolled uptake and replication of Mtb. Furthermore, we provide evidence that this pathway is critical for mycobacterial entry, while the cellular infection with other pathogens, such as Shigella flexneri and Yersinia pseudotuberculosis, is not affected. Altogether, these results reveal how cortical actin plays the role of a barrier to prevent mycobacterial entry into epithelial cells and indicate a novel role for ArfGAP1 as a restriction factor of host–pathogen interactions.