INTRODUCTION

TOP ABSTRACT INTRODUCTION MICROFILAMENTS MICROTUBULES INTERMEDIATE FILAMENTS CONCLUDING REMARKS REFERENCES

THIS REVIEW FOCUSES ON THE three major cytoskeletal protein families in epithelial cells of the digestive system, including the liver, pancreas, esophagus, stomach, intestine, and gallbladder. The term cytoskeleton derives from "cyto" (kutos in Greek meaning "hollow vessel") and "skeleton" (skeletos in Greek meaning "dried up"). Cytoskeleton refers to the major fibrillar elements that are found in cells if one removes the soluble cytosol and intracellular organelles. The three major protein families of the cytoskeleton fibrillar systems are microfilaments (MF), microtubules (MT), and intermediate filaments (IF) (Table 1), which in turn interact with a growing list of associated proteins. Although excellent reviews have addressed MF, MT, or IF individually, we are not aware of any review that addresses and contrasts side by side the three major cytoskeletal protein groups within digestive epithelia.

Several cell types develop a polarized architecture that is essential for their biological function. Typical polarized cells are neurons and epithelial cells, which partake in endocytosis, exocytosis, and vesicle transport. In epithelial cells, the apical surface faces the lumen or canaliculus and is the site of secretion or absorption. The basolateral surfaces refer to basal areas, which contain hemidesmosomes and interact with the extracellular matrix (ECM), and the lateral sides, which interconnect neighboring cells via gap junctions and desmosomes (Fig. 1). Secretory and membrane proteins are transported in membrane-bound vesicles from the endoplasmic reticulum (ER) to the Golgi network and then are sorted in the trans-Golgi network (TGN) to the plasma membrane or other organelles. Basolateral sorting is mediated by signals (such as specific amino acid sequences) that reside within the protein to be transported (e.g., see Ref. 120). There are also apical signals (such as N glycosylation and glycosylphosphatidylinositol anchor) and late endosome sorting signals (120). Hepatocytes appear to be unique among polarized cells in that most apical transport of proteins occurs indirectly via initial transport to the basolateral membrane followed by transcytosis, except for the direct apical transport of sphingolipids (277).

View larger version (96K): [in this window] [in a new window]   Fig. 1.   Schematic diagram of epithelial cell polarity, cell-cell, and cell-substratum junctions. Polarized epithelia, as found in the intestine, contain an apical domain with specialized features such as microvilli and a basolateral domain that are separated by tight junctions. Plasma membrane proteins reach their ultimate target domain by a direct or an indirect (transcytotic) pathway involving microfilaments (MF) and microtubules (MT). The apical and basolateral domains have distinct organization of underlying cytoskeleton. For example, the MT organizing center (MTOC) underneath the apical membrane generates a uniform polarity of MT with the apical minus and basal plus ends, allowing vesicle transport in two directions. Tight, gap, and adherens junctions and focal contacts link with actin filaments (attachment of MF with gap junctions is not well defined, which is indicated by the close proximity but not attachment to MF), whereas desmosomes and hemidesmosomes connect with intermediate filaments (IF). A, apical; BL, basolateral; T, tight junction; AJ, adherens junction; D, desmosome; G, gap junction; HD, hemidesmosome; FC, focal contacts; E, endocytosis; TC, transcytosis; N, nucleus; ER, endoplasmic reticulum.

The three major activities of digestive epithelial cells, namely, secretion, digestion, and absorption, require the establishment and maintenance of cellular polarity and intracellular transport, all functions intimately linked to the cytoskeleton. Other important roles for the cytoskeleton in digestive and nondigestive-type epithelia include its involvement in mitosis, protection from environmental stresses, cell and intracellular organelle anchorage, gene regulation, and motility during migration, differentiation, and wound repair. In addition, a new role for the cytoskeleton in signal transduction is emerging, as contrasted with the well-established reorganization of the cytoskeleton on signaling, since many signaling molecules associate with cytoskeletal proteins.

Although the three major cytoskeletal proteins do share some functions, which likely provide an important functional redundancy mechanism for cells, they also have several distinct properties (Table 1). For example, the diverse structure of IF proteins and their selective expression in higher eukaryotes imply one or more specialized evolutionary roles, which appear to include a protective role from a wide range of environmental stresses. In addition, some of the properties of IF proteins, compared with MF and MT, have made their study very difficult. For example, the absence of cytoplasmic IF proteins from yeast and Drosophila (which precluded functional genetic studies), their relative insolubility (which made biochemical separation for dynamic studies difficult), and the lack of selective stabilizers and destabilizers have contributed to the difficulty in appreciating their biological significance. Although many diseases result in modulation of all three cytoskeletal protein families, it is mainly mutations in IF proteins, and to a lesser extent actin, that are known to directly result in several human diseases. As our understanding of the cytoskeleton advances, it is hoped that targeted manipulation of cytoskeletal components will offer novel and effective therapeutic advantages. This review will highlight general physiological features of the three major cytoskeletal protein families and summarize their direct and indirect association with disease states of the digestive tract.

	MICROFILAMENTS

TOP ABSTRACT INTRODUCTION MICROFILAMENTS MICROTUBULES INTERMEDIATE FILAMENTS CONCLUDING REMARKS REFERENCES

Actin and Regulation of Actin Dynamics

Actin, actin-binding, actin-related proteins, and their regulation. The actin cytoskeleton is highly conserved in all eukaryotic cells, from protozoa to yeast to human, and is composed of actin as its major component and actin-binding proteins (220). The human actin gene family includes three classes, named -, -, and -actin. There are six different actin genes in mammals, two ubiquitous nonmuscle ( and ), two striated muscle [₁ (skeletal) and  (cardiac)], and two smooth muscle (₂ or  of vascular smooth muscle and ₃ or  of enteric smooth muscle) isoforms (220). The amino acid sequences of these three gene product classes are almost identical (molecular mass of ~42 kDa), with 93-98% sequence identity between the -, , and -isoforms. MF are formed by self-assembly of G actin and are decorated with actin-binding proteins. Polymeric actins (F actin) are further assembled into a filamentous network, with regulation by a large number of actin-binding proteins, which, in turn, are regulated by extracellular or intracellular signals.

Actin cytoskeleton assembly and its organization. MF are 5- to 8-nm-wide helixes of uniformly oriented G actin. The two ends of the filament polymerize at different rates, such that the rate of the fast-growing "barbed end" is 20 times that of the opposing "pointed end." In nonmuscle cells, ~50% of actin is filamentous and the remaining is monomeric. MF are highly dynamic, with a half time of minutes as determined by video microscopy of a GFP-actin fusion protein in cultured cells (34). Actin assembly and disassembly are regulated, in response to stimuli or during the cell cycle, through one or more of many actin-binding proteins, which function in filament nucleation (the rate-limiting step), anchoring, capping, severing, cross-linking, and bundling (Fig. 2). MF are nucleated at multiple sites beneath the plasma membrane, in association with the Arp2-Arp3 complex (Fig. 2) and in proximity to focal adhesions, AJ, or tight junctions (TJ) (Fig. 1).

View larger version (88K): [in this window] [in a new window]   Fig. 2.   Formation of an orthogonal actin filament network in lamellipodia. 1) Cdc42 is activated and binds to the plasma membrane, where signals are received to form a lamellipodium, and then recruits N-Wiskott-Aldrich syndrome protein (WASP), which is targeted to the leading edge membrane through binding to focal contact component vinculin. 2) Cdc42-activated N-WASP induces Arp2 and Arp3 realignment to present a surface that is similar to the barbed end of an actin filament, thereby promoting actin filament nucleation. 3) MF are elongated by the addition of ATP-bound actin monomer in the barbed end. Elongation rate can be regulated by Ena/vasodilator-stimulated phosphoproteins. 4) Scar binds to the side of actin filaments and recruits Arp2/3 complex, which nucleates new actin filaments with free barbed ends to form a branched network and then results in the protrusion of lamellipodia. 5) MF are capped in barbed ends by capping protein and gelsolin to prevent the addition of actin monomers. Dynamically capping and uncapping can be controlled by signaling intermediates such as plasma membrane phosphoinositides. 6) Actin-bound ATP is hydrolyzed to ADP by the actin intrinsic ATPase activity and then actin-severing proteins such as cofilin bind to ADP-bound actin and result in MF disassembly mainly in regions toward the cell body. 7) Released ADP-actin monomer is sequestered by profilin and exchanges its nucleotide to ATP. ATP-actin-profilin is then ready for polymerization into new actin filaments (see Ref. 152 and references therein).

Myosin-based force transduction. MF provide tracks for ATP-driven myosin motor proteins, which translocate along actin to generate forces necessary for contraction, vesicle trafficking, organelle localization, and signal transduction. There are at least 14 distinct classes of myosins (164), of which myosin I and V subfamilies (MyoI or MyoVa) play a role in vesicle/particle transport. As measured by an optical tweezer transducer, the chick intestinal apical MyoI moves along MF 11.5 nm in two steps during one ATP hydrolysis cycle (251). MyoVa interacts directly with the MT-based kinesin isoform KhcU, which suggests that cellular transport may be coordinated through the direct interaction of different motor molecules on different polymer tracks. Current evidence suggests that MF and MT systems cooperate such that the long-range transport of cellular components in animal cells is based on MT networks, whereas the actin network appears to be critical for short-range transport (95). In support of this, MT do not extend to the cell periphery, whereas MF tend to be organized preferentially in cortical regions of the cell. Also, cytoplasmic vesicles from squid axoplasm translocate along both MF and MT, and a single vesicle is found to switch from MT to MF and vice versa. Thus one proposed model is that secretory vesicles budding off the Golgi complex travel to their destination at the cell periphery via long-range movements carried out by MT motor proteins along MT and then switch "track" to MF to traverse the actin-rich cell cortex with the help of myosins (10).

Function of the Actin Cytoskeleton in Digestive Epithelia

Cytoplasmic organization, cell shape, and polarity. The cytoplasm of a eukaryotic cell is spatially and temporally organized by MF, MT, IF, and their associated proteins, which form latticelike mesh networks to restrict free diffusion of molecules larger than 500 kDa (149, 217). Distinct populations of actin filaments are associated with different cellular compartments, e.g., terminal web, stress fibers, filopodia, and lamellipodia. These specific actin compartments consist of different isoforms, which in turn can associate with unique actin-binding proteins in an actin isoform-specific fashion. For example, - and -actins are differentially distributed in gastric parietal cells (269). The -actin isoform is found along the entire gland lumen and associates preferentially with ezrin in gastric epithelial cells, whereas the -actin isoform is distributed preferentially near the basolateral membrane. In general, -actin is present in most, if not all, nonmuscle MF, whereas -actin is enriched in cytoplasmic and membrane structures undergoing or capable of undergoing rapid remodeling or polarized movement (81). MF provide a large charged surface area for potential localization of cytoplasmic components (103). This scaffolding function is supported by the fact that there is a 47,000-µm² surface area on MF in a 20-µm-diameter cell with a typical concentration of 10 mg/ml F actin, compared with only 700-µm² plasma membrane surface area. The MF-dependent localization of cellular components includes glycolytic enzymes, Src tyrosine kinase and PKC, organelles, and mRNA. Thus the actin cytoskeleton, in cooperation with MT and IF, provides a three-dimensional framework to structure the cytoplasm and to compartmentalize cellular events.

Cell-cell and cell-matrix interaction. Epithelial cells form highly specialized actin-anchoring membrane structures to facilitate cell-cell and cell-ECM contacts, which are likely essential for maintaining cell morphology and tissue integrity. Abnormalities in these contacts are associated with a variety of diseases, including diarrhea, carcinogenesis, and metastasis. All these contacts consist of transmembrane proteins that interact with neighboring cells or with ECM via their extracellular portions and with cytoplasmic adaptor molecules via their intracellular domains. Many of the cytoplasmic adaptor proteins interact with the actin cytoskeleton to strengthen the contacts. Examples of important epithelial actin-associated membrane structures include the following (see Fig. 1): 1) TJ, which cross-link cells to form a "fence" and are the gatekeepers that regulate the paracellular pathway and epithelial permeability. TJ (also called zonula occludens) consist of two families of four-transmembrane domain proteins, namely occludin (71) and claudin (177), and one single transmembrane domain protein, JAM (158), along with associated cytoplasmic proteins, including ZO-1, ZO-2, and ZO-3. The ZO proteins associate with each other via their PDZ domains (named for postsynaptic density protein 95, disks large, ZO-1), bind actin via the ZO-1 proline-rich tail or via -catenin, and bind occludin via their guanylate kinase domains. The organization and function of TJ may be regulated by RhoA and Rac1 through regulation of MF in the apical pole of polarized enterocytes (112, 188). 2) AJ, which include the homotypically binding transmembrane protein E-cadherin that helps form a continuous adhesion belt around each interacting cell in the epithelial sheet, is another important epithelial actin-associated membrane structure. Other AJ cytoplasmic components include the actin-binding proteins vinculin, -catenin, -actinin, paxillin, talin, vasodilator-stimulated phosphoprotein, and adaptor proteins such as vinexin, -catenin, and plakoglobin. -Catenin plays a critical role in the transmembrane anchorage of cadherins, since deletion of -catenin inactivates cadherin-mediated cell adhesion, resulting in a nonadhesive phenotype (99). Through these actin-binding proteins, a contractile bundle of actin-myosin filaments runs along the cytoplasmic surface of the junctional plasma membrane and links cell to cell via E-cadherin to generate an extensive transcellular network. The coordinated contraction of this actin network plays several roles in epithelial cells, including canalicular contraction during bile secretion. 3) Gap junctions, which connect neighboring cells by intercellular channels that consist of connexins, a family of four-transmembrane domain proteins with >14 different genes in mice, is a third important epithelial actin-associated membrane structure. Molecules less than ~1 kDa, including ions, metabolites, and messengers, pass freely through these channels. The role of gap junctions in digestive epithelia is important for coordinated tissue behavior, but their interaction with cytoskeletal elements is not clear. Although connexin mutations cause several human diseases, including nonsyndromic deafness and X-linked Charcot-Marie-Tooth disease, no digestive disease is known to be caused by connexin mutations (221). 4) Focal adhesions (i.e., focal contacts), which connect the actin cytoskeleton to the ECM through the integrins and their associated cytosolic actin-binding proteins, is a fourth important epithelial actin-associated membrane structure. The ectodomain of integrins binds to ECM, whereas their intracellular domain binds to many actin-binding proteins such as talin, vinculin, paxillin, and -actinin, which in turn interact with actin (75).

Cell motility. Cell motility is a tightly integrated process between different components of the cytoskeleton (171). Actin-based cell motility plays important roles in epithelial cell functions, including secretory vesicle movement, cell movement during wound healing, and cell regeneration. Three types of actin-based epithelial cell motility can be categorized as those that 1) utilize already assembled MF as in cytosolic particle transport and during cytokinesis, 2) utilize new actin assembly as occurs during pathogenic intra- and intercellular bacterial movement, and 3) utilize preassembled and new MF assembly as occurs during cell locomotion. Examples of these categories are outlined as follows: 1) for cytokinesis, epithelial cells undergo a dramatic change in morphology during mitosis, which requires reorganization of all three cytoskeletal filaments and concludes with separation of daughter cells via cytokinesis. The cytoplasmic constriction at the equator during dividing cell separation is driven by a force generated by the preassembled contractile ring, which consists of membrane-associated actin and actin-binding proteins such as myosin II and cortexillins (256). This constriction draws the plasma membrane inward to form a "cleavage furrow," which gradually deepens and finally breaks at each end to separate two daughter cells. Other cell-cell contacts are also affected during cytokinesis, such as AJ, which partially lose contact with the actin cytoskeleton. 2) For intracellular movement, Listeria monocytogenes and Shigella flexneri movement is driven by local catalysis of MF polymerization at one side of the bacteria that forms an actin comet tail to drive bacteria forward. Also, endocytic vesicle movement, after pinching off the plasma membrane into the cytosol, is advanced by a brief burst of "mini-comet tail" actin polymerization (165). 3) For locomotion, a moving cell is morphologically polarized and is often described as four cell regions from front to back: lamellipodium in the leading edge, lamella that is located immediately behind the lamellipodium and is thicker, cell body that contains the nucleus and other organelles, and the remaining cell rear (38). The lamellipodium protrudes outward by dynamic actin polymerization forces. The lamella appears to stay in place, whereas the cell body translocates by previously assembled actin-myosin filaments. The formation of an orthogonal MF network in lamellipodia is illustrated in Fig. 2. In some (see Ref. 253) but not all (Ref. 168) systems, MT enter newly formed lamellipodia and generate net plus-end growth that appears to be regulated by actin dynamics, whereas MT breakage and shortening near the cell body at the base of lamella depends on actin-myosin (253). This MT elongation in the lamellipodium locally activates Rac1 (255), and MT disassembling in cell body locally activates RhoA (202). In turn, Rac1 promotes further MF assembly (i.e., dynamic component) at the leading edge (via Scar1 and Arp2-Arp3 complex pathway activation), which enhances MT growth in the lamellipodium, whereas RhoA drives actin-myosin contractility (via a preassembled component) and formation of focal adhesions in the cell body and near the cell edge, which provide the base for cytoplasmic movement (254).

Secretion and absorption. The actin cytoskeleton is reorganized during epithelial cell secretion, as in exocytosis and transcytosis, to regulate vesicle transport in response to intra- and extracellular signals (163). The cell apex actin terminal web (Fig. 1) may provide a physical structure to anchor secretory vesicles in proximity to the plasma membrane. On stimulation, an actin-myosin network (including profilin, ADF/cofilin, and capping protein) reorganizes to release the physical barrier for granules and to allow movement to membrane docking sites (245). For example, stimulated acid secretion from parietal cells is inhibited by cytochalasins in a dose-dependent fashion (63), presumably by disrupting the cortex actin "tracks" for vesicle transport. Several vesicle transport-related myosins are found apically in enterocytes, hepatocytes, and pancreatic acinar cells. For example, MyoI binds to nascent, post-Golgi secretory vesicles that are transported to the enterocyte apex (222). Digestive epithelial cells can also transport nonpermeable molecules and particles from the apical to the basolateral membrane, or vice versa, via transcytosis (Fig. 1). It is assumed that MF plays a similar role in transcytosis, as it does in exocytosis and endocytosis, based in part on cytochalasin D inhibition of basolateral to apical transcytosis of sphingolipids in polarized Hep G2 cells (276). In addition, luminal secretion of electrolytes to maintain intestinal content fluidity and mucosal defense is tightly regulated by ion channels and TJ-gated paracellular pathways, which also play a role in absorption (13).

Signal transduction. The effect of cytochalasin treatment of cells on signal transduction and the colocalization of many signaling molecules with MF on activation of specific signaling pathways provide strong evidence for MF modulation of cell signaling (103). Examples include integrin-mediated mechanical stress induction of mRNA and ribosomal movement in an actin-dependent fashion (32), T cell receptor -chain tyrosine phosphorylation-triggered binding to MF (207), and actin treadmilling regulation of the serum response factor activation (224). Also, stimulation of many protein and lipid kinases and GTPases induces their translocation to MF, thereby suggesting a scaffolding, regulation of availability, or compartmentalization function for MF (103). For example, c-Src tyrosine kinase localizes to MF in Rous sarcoma virus-infected cells, presumably through binding to vinculin. Furthermore, cytochalasin-induced disruption of MF blocks c-Src localization to the cell periphery in fibroblasts, whereas the MT destabilizing drug nocodazole has no effect on Src translocation. Signals that stimulate Src translocation to MF include platelet-derived growth factor, epidermal growth factor, thrombin, and RhoA. Moreover, PKC- and -, phospholipase C-, duet serine/threonine kinase, Rho, Rac, and Cdc42 associate with MF with presumed functional implications (103).

Actin Cytoskeleton in Digestive Diseases

Infection. A variety of microbes infect digestive epithelial cells or affect them indirectly via toxins, with dramatic reorganization of MF (61). For example, enteropathogenic Escherichia coli (EPEC) attaches on the epithelial cell membrane and then secretes and translocates a membrane-bound protein, Tir (translocated intimin receptor), into the host cell membrane via a type III secretion system (72). The translocated extracellular domain of Tir binds to the EPEC surface protein intimin- to anchor the bacteria to the epithelial cell membrane, whereas its cytosolic domain becomes tyrosine phosphorylated and recruits CHP (a Cdc42 homologous protein, Ref. 6). Then, CHP recruits N-WASP to initiate Arp2/3 complex-dependent actin polymerization. These EPEC-linked MF can extend up to 10 µm in length, are resistant to cytochalasin D, and form a pedestal-like structure to hold EPEC on the epithelial membrane and subsequently result in diarrhea. Similarly, Helicobacter pylori colonizes gastric surface cells and alters host cell actin organization to form a pedestal structure (37). Although the pathogen-induced changes in MF may play an important role in propagation of a given pathogen, it is unclear if these changes play a direct role in diarrheal secretion per se.

Cancer. The involvement of MF in cancer development and/or progression is likely to be indirect but nevertheless important. For example, loss of MF correlated with the transition of human noninvasive benign colonic tumors into invasive malignancies (64). The suppression of migration and proliferation of a human gastric cancer cell line by MF disruption suggests that there may be an association between changes in MF and gastric cancer metastasis in this cell line model (102). In addition, the actin-binding protein merlin functions as a tumor suppressor in schwannomas and meningiomas (82), but the role of merlin or other actin- binding proteins in epithelial cancers is unknown. Of note, wild-type p53 binds directly to F actin with a dissociation constant of ~10 µM (166), but the role of this interaction in tumor biology is not known.

Inflammation. MF and its associated cell-cell interactions are involved in intestinal inflammation. For example, rearrangement of the epithelial cortical MF network in T84 cells is accompanied by polymorphonuclear leukocyte transepithelial migration in a physiological basolateral-to-apical direction or in the reverse direction. However, pretreatment of cells with the F actin-stabilizing agent phallacidin greatly enhanced migration in the reverse direction, which suggests that the epithelial actin rearrangement can selectively affect polymorphonuclear leukocyte migration (91). Also, E-cadherin expression and -catenin expression during mucosal ulceration in inflammatory bowel disease decrease, which may promote cell migration during epithelial restitution of the gastrointestinal mucosa (116). E-cadherin/-catenin complexes also rapidly dissociate in acute pancreatitis (145). In addition, TJ are disrupted in human intestinal HT-29 cells by tumor necrosis factor-, with impairment of barrier function, which may play a pathogenic role in intestinal inflammation (214). The intestinal barrier function is also disrupted in rat experimental colitis in association with secretion of rat mast cell protease II, which may contribute to the pathogenesis of intestinal inflammation (225).

Apoptosis. Apoptotic cells undergo dramatic morphological changes to generate apoptotic bodies, the occurrence of which requires cytoskeletal reorganization. It is, however, unclear if cytoskeletal alterations during apoptosis are an epiphenomenon or if they play any direct role in the progression of apoptosis. Notably, actin and actin-binding proteins such as gelsolin and fodrin are caspase substrates in several tumor cell line apoptosis models (e.g., see Refs. 119 and 133). For example, caspase-3-dependent actin cleavage generates two fragments (15 and 31 kDa) in human tumor cell lines undergoing apoptosis, including HeLa (epitheloid), A431 (epidermoid), and U-937 (myeloid), presumably at the predicted Asp-244. Ectopic expression of this apoptotic 15-kDa actin fragment in human embryonic kidney 293T cells induces morphological changes of actin localization resembling those of apoptotic cells independent of caspase activation (159). However, apoptosis-associated actin cleavage was not found in U937, HeLa, or human Burkitt lymphoma cell lines in an independent study (223). Hence, apoptosis-associated cleavage of actin and/or its binding proteins appears to depend on the system studied, but evidence to date supports an important role for actin/actin-binding protein reorganization and/or degradation during apoptosis.

Liver disease. In acute liver injury, there is expansion of the stellate cell population in conjunction with smooth muscle actin expression. During chronic liver injury, the stellate cell differentiates into a myofibroblast-like cell, which has a high fibrogenic capacity and is involved in ECM degradation (65). Analysis of smooth muscle actin from liver biopsies can identify the myofibroblastic transformation in injured liver, whereby the extent of smooth muscle actin-positive cells reflects progression to hepatic fibrosis and cirrhosis. In addition, autoantibodies against actin are found in association with autoimmune chronic liver disease and in chronic hepatitis C (27). Anti-actin antibodies are at least one of the components of the so-called anti-smooth muscle antibodies, and their presence may help differentiate hepatitis C-related vs. nonhepatitis C-related autoimmune liver disease (27). Presence of anti-actin antibodies may also correlate with a poorer prognosis and with HLA-DR3 expression (43). Other potentially useful serological markers include serum secretory gelsolin, which was decreased in patients with acute liver failure (228). However, the pathophysiological role of actin and its binding proteins in liver diseases is not known.

	MICROTUBULES

TOP ABSTRACT INTRODUCTION MICROFILAMENTS MICROTUBULES INTERMEDIATE FILAMENTS CONCLUDING REMARKS REFERENCES

Overview of MT

Both - and -tubulin bind GTP, and, shortly after subunit assembly, the -tubulin-bound GTP is hydrolyzed to release one phosphate followed by addition of an -dimer to the MT end (48). Tubulin dimers are added to the MT from head to tail, and the two MT ends are distinct in their polymerization rates in that one is faster growing (plus end) than the other (minus end). This polarity has very important physiological ramifications, including determining the directionality of cargo transport by motor proteins along the MT. The -heterodimers assemble into linear protofilaments such that dimers are oriented with -tubulin toward the plus end and -tubulin toward the minus end of the MT (48). In vivo, MT consist predominantly of 13 protofilaments. Detailed MT architecture was recently described based on the tertiary structure of tubulin and a 20 Å resolution map of the MT (186).

MT dynamics are governed primarily by a mechanism called "dynamic instability," postulating that a single MT never reaches a steady-state length but persists in prolonged states of polymerization and depolymerization. In the dynamic instability model, MT stability is thought to depend on a -tubulin-GTP-cap at the MT plus end where hydrolysis has not yet occurred (48). MT stability is also highly regulated by several binding proteins (Table 3). Each MT is oriented with the minus end at the nucleating site and the rapidly changing plus end free in the cytoplasm (48). Nucleation of most MT occurs from the MTOC (also called "centrosome" in animal cells), which is structurally diverse in different cell types but shares a common ability to nucleate MT (111). In nonpolarized cells, the MTOC is positioned at the cell center, to one side of the nucleus, whereas in polarized epithelial cells MT nucleation appears to occur from several noncentrosome-dependent sites near the apical membrane (83, 163). For example, hepatocyte MT minus ends originate from several organizing centers positioned at the apical pericanalicular region (187) and centrosomal structures are found just below the apical membrane in enterocytes and in the human intestinal cell line Caco-2 (83). Hence, due to the apical nucleation, most minus ends of MT in all studied polarized digestive epithelia face the apical area and the plus ends extend through the cell body to the basolateral surface (Fig. 1, also see General directionality issues that pertain to MT function below). This polarized distribution was demonstrated in Caco-2, WIF-B (liver), and MDCK cells using a MT hook decoration assay and electron microscopy and is also likely present in enterocytes and pancreatic acinar cells (83, 163). Hepatocytes have, in addition to the apical to basal MT, some "curved" MT that intersect the "straight" MT, and it is hypothesized that the curved MT define TGN areas (83).

MT Binding Proteins

MT motor proteins. Given the secretory and absorptive features of digestive epithelia, the molecular motors of MT, namely kinesin and cytoplasmic dynein and their superfamilies, are particularly important for moving cargo (83, 163). Both motor families utilize ATP hydrolysis to power vesicle movements along MT tracks (90, 163). Kinesins make up a large protein family that is important in force-generated cellular activities such as chromosomal segregation, protein and vesicle transport, and intracellular organization (Ref. 90, also see http://blocks.fhcrc.org/kinesin). The location of the structural motor domain neck region of kinesin heavy chain divides kinesins into 1) Kin N, 2) Kin C, and 3) Kin I motors (i.e., neck domain toward the NH₂ terminus, COOH terminus, or interior of the molecule, respectively). Kin N motors (e.g., conventional kinesin) and Kin I are MT plus-end directed (anterograde transport), whereas Kin C motors are MT minus-end (retrograde) directed. The kinesin structural diversities form the basis for specialized transport of various cargoes (163). Our understanding of kinesins comes primarily from yeast and Drosophila studies, but a few reports have described kinesins in digestive epithelia, including rat pancreas (e.g., Ref. 157) and liver (130).

MAP and destabilizing effectors. MAP were initially identified by virtue of their copurification with MT and were shown to promote tubulin polymerization and to stabilize MT (54). For example, MAP overexpression inhibits vesicle transport and organelle movement and stabilizes and bundles MT. MAP2C and tau also act by virtue of their extension as a projection domain away from MT and as such can bundle MT and act as a scaffold for signaling elements or organelles. For example, several signaling molecules associate with MAP, such as protein kinase A, which binds to MAP2, and cyclin B, which binds to MAP4 (Ref. 80 and Table 3). Phosphorylation plays an important role in regulating MAP and usually weakens MAP-MT binding with subsequent MT destabilization on release of MAP. Some kinases may also be classified as MAP, as exemplified by the MT affinity-regulating kinases, which phosphorylate MAP and lead to increased MT turnover and perhaps facilitated transport along MT (54).

Function of MT in Digestive Epithelia

General directionality issues that pertain to MT function. In polarized digestive epithelial cells, MT and their associated motor proteins are particularly important in facilitating and organizing vesicle transport during endocytosis and exocytosis (83, 163). Confusion may occur due to fundamental differences in MT organization between polarized and nonpolarized cells, which are based on the different localizations of the MTOC. For example, the vast amount of MT minus ends in most nonpolarized cells is found at the perinuclear MTOC, whereas the plus ends radiate out to all sections of the plasma membrane. This implies that exocytosis in nonpolarized cells is always MT plus-end directed, whereas endocytosis is minus-end directed. In contrast, polarized epithelial cell MT are generally organized with their minus ends at the cell apex, indicating that apical exocytosis is expected to be MT minus-end directed and endocytosis from the apical regions (as in enterocytes) is expected to be plus-end directed. It should also not be overlooked that, although most MT are positioned with the minus ends at the apical region in polarized epithelial cells, there is a pool of MT that runs in the opposite direction (9). A further variation of this organization is found in neurons, which also are polarized cells, but in contrast to polarized epithelial cells neurons have their MTOC located in the cell body and the plus end in the axon, where secretion of synaptic vesicles occurs (8). These differences caution against generalizing MT-mediated transport results from different cell types. Attempts at unifying MT directionality with function are further complicated by the position of the Golgi and ER relative to MT in different cells. For example, hepatocyte Golgi are situated at the apical canalicular membrane at the MT minus ends, whereas pancreatic acinar cell and enterocyte Golgi are located supranuclearly at the MT plus ends (163). These differences may reflect the use of different motor proteins in different cells and/or the use of kinesins that can be plus or minus end directed.

Cell polarity and organelle positioning. Although MT do not directly interact with cell junctions as MF and IF do, they reorganize on cell junction formation with the aid of >50 MT-regulating proteins (80, 83). However, the functional significance of such reorganization is poorly understood. In contrast, MT and their motors are very important in organizing and positioning organelles, including Golgi, ER, endosomes, lysosomes, peroxisomes, and chromosomes, during mitosis (90, 111, 235). The importance of MT in Golgi positioning is supported by Golgi redistribution to peripheral cellular sites in the presence of MT-disrupting agents and the identification of a Golgi MT-associated protein (GMAP-210) that links cis-Golgi to MT minus ends in HeLa cells (100). Cytoplasmic dynein is also important for Golgi and vesicle positioning, in part because dynein-null murine blastocysts contain a highly vesiculated and redistributed Golgi complex throughout the cytoplasm with redistributed endosomes and lysosomes (84). Golgi membranes also associate with kinesin in several cell types, including rat hepatocytes (163). The reason for this association is unclear but could reflect a role for kinesin in plus-end MT-directed recycling of membranes back to the ER.

Absorption and endocytosis. Transport of endocytic vesicles from the basolateral plasma membrane of polarized epithelial cells is MT based and involves dynein (83). For example, hepatocyte dynein-driven receptor-mediated basolateral endocytosis of asialoglycoproteins (ASGP) follows the sequence of ASGP binding to their membrane receptor and then localizing in endosomal vesicles in association with MT. It appears that only ASGP, but not their receptors, bind to MT via dynein followed by transport toward pericentrosomal lysosomes (78, 83, 163). In other systems, MT also play a role in membrane and receptor recycling. For example, the apical pericentriolar endosomal compartment in Caco-2 or MDCK cells sorts membranes back to their site of origin after internalization (129) in a MT-dependent fashion using the small GTPases Rab25 and Rab11a (26). Rab proteins also associate with pancreatic zymogen granules (190), gastric parietal secretory granules (24), and hepatocyte transcytotic vesicles (107). The roles of MT and their motors in apical endocytosis are still unclear, although it appears that polymerized MT are needed for apical endocytosis and for the convergence of both the apical and basolateral endocytosis pathways, which occurs at the level of late endosomes. In addition, the nonmotor protein, 170-kDa cytoplasmic linker protein (CLIP-170), may play a role, since it links endocytic vesicles to MT by binding MT plus ends in transfected cells (50). Other potential modes of regulating endocytosis includes MT phosphorylation, given that MT are substrates of G protein-coupled receptor kinase, which has a role in downregulating receptor signaling by promoting receptor endocytosis (80).

Secretion and exocytosis. Secretory proteins are transported from ER to Golgi and further packed into distinct vesicles that are destined for the apical or basolateral membrane compartments. Most data suggest that MT and MT motors facilitate the transport of apical proteins but are less involved in the transport of basolaterally destined proteins. MT involvement in these processes is based almost exclusively on studies using MT-disrupting drugs with resultant missorting of apical (e.g., gp80 glycoprotein) and basolateral (e.g., Ag525, albumin) proteins in hepatocytes or enterocytes (76, 83, 162). In contrast, Ig receptor and Na⁺-K⁺-ATPase transport to the basolateral membrane is not significantly affected by nocodazole in MDCK and intestinal cells, thereby supporting a minimal MT role in basolateral transport (22, 83). As for transcytosis, which is highly relevant in digestive epithelia, there is evidence for MT-dependent and -independent transcytotic transport (83, 276).

Mitosis and development. The classical and most dramatic reorganization of MT occurs during mitosis when MT help segregate the duplicated chromosomes to the two daughter cells. Although MT dynamics during mitosis have not been appreciably studied in digestive epithelia, their implications on such epithelia are clear during normal and abnormal cell division, as noted in regenerating hepatocytes (e.g., after partial hepatectomy) and enterocytes and during cancerous growth, respectively. The interphase radial array of MT changes during mitosis into bipolar MT arrays, which originate from the duplicated centrosomes and interact with the condensed chromosomes at the kinetochores (111). Most of the cell cycle-dependent regulation of MT assembly occurs in association with, and is likely due to, changes in phosphorylation of MT accessory proteins. For example, the MT destabilizer Op18 requires phosphorylation on four serines for mitotic spindle formation (28). Op18 expression also increases after partial hepatectomy (131) in association with hepatocyte MT depolymerization (19). In addition, cytoplasmic dynein is indispensable during mitosis due to its multiple roles in assembling and stabilizing the spindle pole, so that centrosomes, together with dynein and dynactin, play a cooperative role in chromosome alignment during mitosis (90, 117).

Signal transduction. The enormous MT protein surface area and their organization in a polarized fashion from the apical membrane to the nucleus make them excellent candidates to regulate intracellular signaling events (80, 103). This is supported by the increasing number of characterized signaling proteins, including phosphatases, kinases, transcription factors, and adaptor molecules that interact with MT, MT motors, or MAP (Table 3). In most cases, the significance of MT-signaling protein interactions is not known, since the interacting domains have not been characterized to allow functional studies. Three types of MT signaling factor interactions can be envisioned. 1) The first is direct interaction with the MT. An example is the small GTPase Rac, which binds MT in vitro and colocalizes with MT in vivo. 2) The second is indirect interaction via a motor protein, as for the physical interaction of the mixed-lineage kinase (MLK2, an activator of the Jun kinase pathway) with the kinesin-like KIF3 family. 3) The third is indirect interaction via a nonmotor protein, such as protein kinase A binding with MAP2 and cyclin B binding with MAP4 (80). MT direct and indirect interactions with signaling molecules suggest that MT may act as a scaffold that brings together components of signaling pathways, thereby regulating their molecular availability (80). Although there are few MT-related signaling studies in digestive epithelia, there are some examples of changes in MT dynamics in association with alterations of cell protein phosphorylation, as noted in hepatocyte growth factor upregulation of kinesin, myosin, and tubulin gene expression in hepatocytes (240).

Digestive Disease Association of MT and Their Effector Proteins

Liver diseases. Ethanol exposure affects several hepatic MT-regulated processes and is associated with decreased receptor-mediated endocytosis and biliary secretion and accumulation of proteins in the Golgi apparatus (83). Chronic alcohol consumption reduces MT in human hepatocytes (160), possibly due to interference with the ability of tubulin to polymerize when isolated in vitro from ethanol-treated rats (273). The effect of alcoholic liver disease on MT and their assembly competence may relate to the high affinity of acetaldehyde (an ethanol oxidation product) to -tubulin, particularly since acetaldehyde-conjugated -tubulin inhibits tubulin assembly into MT (83). Hence, the reduced vesicle movement in hepatocytes when subjected to alcohol exposure may be due to direct effects on MT or may be indirect such as via motor function impairment, as noted by the reduction of dynamin association with Golgi membranes (83). Impairment of MT functions has also been observed in intrahepatic cholestasis. For example, cholestatic concentrations of chenodeoxycholate conjugates inhibit the activity of MT motors, thereby suggesting a possible mechanism for vesicular transport impairment in cholestasis (83). In addition, antibodies to MT have been observed in sera from patients with alcoholic liver disease (143) and hepatitis delta (275) and hepatitis B (148) virus infections. The biological significance of tubulin autoantibodies and their clinical utility, if any, are unclear.

Pancreatitis. Caerulein-induced pancreatitis is an established experimental animal model that mimics some of the serological and histological events of human acute pancreatitis. Exposure of animals or isolated acinar cells to caerulein leads to MT disassembly (114, 243) and to increased amylase release (243), thus suggesting a role for MT in pancreatitis. However, both MF (Ref. 114 and D. M. Toivola, N.-O Ku, N. Ghori, S. A. Michie, and M. B. Omary, unpublished observations) and IF are altered in ways similar to MT in the pancreatic models. Hence, disruption of the cytoskeleton in general accompanies pancreatitis, and it is unknown if unique etiologies (e.g., alcohol) preferentially target one cytoskeletal group vs. another. Cytoskeleton-related treatment of acute pancreatitis has not been studied using MF- or IF-modulating drugs in humans, but taxol does protect rats from caerulein-induced pancreatitis (243). This raises the question of whether such drugs can be useful in treating human pancreatitis.

Cancer. Familial and sporadic colon cancers are associated with truncation of the COOH terminus of the human tumor suppressor adenomatous polyposis coli (APC). The COOH terminus of APC physically associates with the EB/RP family of MAP, such as EB1 and RP3, and also likely associates directly with MT, but the precise interacting domains have not been well defined (16). Normally, EB1 is localized with cytoplasmic and spindle MT and coprecipitates with dynein, dynamitin, p150, and other members of the dynactin complex (16). Mutated APC cannot bind to EB1, which provides clues regarding loss of cell cycle control. APC also controls the Wnt signaling pathway by binding to -catenin in a complex with glycogen synthase kinase-3 (GSK-3) and axin/conductin (248). This binding leads to -catenin proteolysis via cascades that include GSK-3 phosphorylation of -catenin, subsequent ubiquitination, and then proteasome-mediated degradation (248). Mutated APC in colonic polyps and cancer cannot bind -catenin, which leads to its nuclear accumulation and then to binding to and activation of the transcription factor Tcf-4, thereby leading to constitutive transcription of target genes including myc and cyclin D (248). The association of MT with carcinogenesis, as occurs with APC, and with other MAP including tau, which is overexpressed in pancreatic exocrine tumors (compared with normal cells, Ref. 249), appears to be highly relevant but indirect.

Infection. Infection of epithelial cells with some viruses or bacteria disrupts MT and can also result in utilizing MT or MT motors as highways for intracellular transport. For example, type 1 human immunodeficiency virus causes MT disruption in human colonic HT-29 cells (46). In addition, dynein plays a role in facilitating adenovirus infection in HeLa and TC7 cells (229). MT may also be important for internalization of some bacteria, given that Campylobacter jejuni uptake was blocked in intestinal cells by MT depolymerization (189). Furthermore, polymerized MT are important in E. coli translocation across the intestinal epithelial barrier (41). The role of MT in microbial pathogenesis is likely cell type and pathogen specific, since nocodazole and colchicine had a limited effect on Listeria monocytogenes uptake in Caco-2 cells compared with other cell lines (141).

Potential use of MT-modifying drugs in digestive disease therapy. In general terms, MT-stabilizing (e.g., taxol) and -destabilizing (e.g., the vinca alkaloids vincristine and vinblastine and colchicine) agents have been used as therapy for several human digestive diseases, including cancer, cirrhosis, acute alcoholic hepatitis, primary biliary cirrhosis, and hepatitis B infection. As such, colchicine has well-proven benefits and is routinely used as therapy and/or prophylaxis in several human diseases, including familial Mediterranean fever and gout (15). Similarly, taxol appears to be relatively beneficial in several malignancies, including esophageal (63a) and gastric (122) adenocarcinomas, but on the basis of phase II studies may not be effective in hepatic (30), pancreatic (263), biliary tree (109), and colorectal (96) adenocarcinomas. The in vivo resistance to anti-MT agents, despite high biological activities in tumor cell lines (219), may be related to altered tubulin isotype expression and/or to alteration in the P glycoprotein multidrug resistance phenotype (e.g., Ref. 55). In nonmalignant liver disease, colchicine is not effective against severe acute alcoholic hepatitis (2) and has mixed results against chronic hepatitis B and cirrhosis (e.g., Ref. 62). However, colchicine may play a beneficial therapeutic role in primary biliary cirrhosis, particularly if used in combination therapy (15). Therefore, the use of MT-modulating agents remains an attractive possibility for certain digestive disease states but warrants further mechanistic, design, and optimum targeting studies.

	INTERMEDIATE FILAMENTS

TOP ABSTRACT INTRODUCTION MICROFILAMENTS MICROTUBULES INTERMEDIATE FILAMENTS CONCLUDING REMARKS REFERENCES

Overview of IF Proteins