The structure and organization of the bile canalicular cytoskeleton with special reference to actin and actin-binding proteins

The Structure and Organization of the Bile Canalicular Cytoskeleton With Special Reference to Actin and Actin-Binding Proteins NOBUHIRO TSUKADA, CAMERON A. ACKERLEY, AND M. JAMES PHILLIPS

The distribution of actin filaments and actin-binding proteins in the bile canaliculus (BC) of normal h u m a n hepatocytes was determined as a means of establishing the structure and organization of the BC cytoskeleton. Immunoblots demonstrated that actin, and the aetinbinding proteins, myosin II, tropomyosin, vineulin, aactinin, villin, were present, as were the non-actin-related proteins ~-tubulin, and cytokeratins. Three aetin filament regions were identified: mierovillus core filaments, a membrane-associated mierofilamentous network, and a circumferential pericanalieular actin filament band. Aetin-binding proteins were nonrandomly associated with aetin in these regions. In the case of the pericanalieular band, t her e was also association with the zonula adherens junction. Intermediate filaments inserted into desmosomes. The ultrastructural localization of the actin-binding proteins was fundamentally linked to the a r r a n g e m e n t and organization of the major canalieulus-associated microfilament structures. Structural organization of the eytoskeleton was also linked to distinct c o m p o n e n t s of the intercellular junctions. It is notable th at tropomyosin and a-actinin, which in muscle cells are regulatory proteins of contractile activity, and myosin II are associated with the pericanalicular actin microfilament band; it is the BC c o u n t e r p a r t of the contractile actin filament band found in the apical region of other secretory cells. The outer sheath of noncontractile intermediate filaments likely stabilizes the eanalicular compartment. ( H E P A T O L O G Y 1995;21:1106-1113.) The bile canaliculus (BC) is the smallest intrahepatic secretory channel of the liver; it plays a central role in hepatic bile s e c r e t i o n ) The canaliculus is formed by a modification of the apical region of the pl as ma membranes of contiguous hepatocytes. The canalicular

Abbreviations: BC, bile canaliculus; SDS PAGE, sodium dodecyl sulfate polyacrylamide gel electrophoresis. From the Research Institute and Department of Pathology, Hospital for Sick Children, and the University of Toronto, Toronto, Ontario, Canada. Received April 13, 1994; accepted November 3, 1994. Supported by the Medical Research Council of Canada, Ottawa, OntariO, Canada (MRC, PPG no. 11810). Address reprint requests to: M. James Phillips, MD, Research Institute and Department of Pathology, Room 3125, Hospital for Sick Children, Toronto, Ontario, Canada M5G 1X8. Copyright © 1995 by the American Association for the Study of Liver Diseases. 0270-9139/95/2104-003253.00/0

m e m b r a n e itself is surrounded by a microfilamentous web of filaments (ectoplasmic zone), which constitutes the subject of this report. The plasma m e m b r a n e of the pericanalicular region is also the site at which liver cells are bound together by the junctional complexes. 2'3 In this report, we have examined the components and organization of the canaliculus-associated cytoskeleton with particular reference to actin-binding proteins. Isolated bile canalicular m e m b r a n e s were subject to sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS PAGE) and immunoblotting techniques to establish which actin-associated proteins were present, and t h e n their distribution in relation to the BC and the contiguous junctional complexes was determined. The localization of the proteins was performed by electron microscopic immunogold localization using chemically fixed Lowicryl-embedded or fixed cryosectioned preparations. The results of this study have been reported in abstract. 4 MATERIALS A N D METHODS Immunob

l o t t i n g ...............

Bile canalicular membranes with adjoining cell junctions and cytoskeletal elements were isolated from human liver tissue (obtained from reduction hepatectomy specimens at the time of segmental liver transplantation) using the method described by Tsukada and Phillips. 5 Briefly, the liver tissue was homogenized in 50 mmol/L KC1, 5 mmol/L MgC12, 1 mmo]/L ethylene glycol bis(~-aminoethyl ether) N,N,N',N'tetraacetic acid, and 25 mmol/L piperazine-N,N'-bis(2-ethanesulfonic acid) (pH 6.9) containing phenyl methyl sulphonyl fluoride. It was then separated by sucrose gradient centrifugation and the fraction collected at a sedimentation coefficient within the range of 1.16 to 1.18 S. Total protein concentration was measured before immunoblotting. For immunoblotting, isolated BC membranes were dissolved in Laemmli SDS sample buffer,6 heated, and applied to a 4% polyacrylamide stacking gel. A 10-mg aliquot of total protein was applied to each lane and to control molecular weight standards (Bio-Rad Laboratories, Inc., Mississauga, Ontario, Canada): Lysates and molecular weight standards were separated by a 5% to 15% linear gradient SDS PAGE. The separated proteins were transferred onto nitrocellulose membrane as described by Towbin et al. 7 After transfer, the total composition of the proteins transferred was determined by Amido black staining. The remaining portions of the membrane were blocked with 5% Carnation instant skim milk

1106

HEPATOLOGYV01. 21, No. 4, 1995 powder before incubation with primary antibody and staining with immunoperoxidase. The commercially prepared primary antibodies used included monoclonal antibodies to chicken gizzard actin (Amersham), tropomyosin (Sigma), vinculin (Sigma), bovine mammary gland epithelium a-actinin (Sigma), cytokeratin (Becton Dickinson), chick brain /~-tubulin (Sigma), villin (Serotec), and a polyclonal antibody to human blood platelet myosin II (Biomedical Laboratories).

TSUKADA, ACKERLEY, AND PHILLIPS 1107

200kD~,--

130kD,,,,100kDPb-

Electron Microscopy of Isolated BC Membranes Representative samples of the isolated BC membranes were fixed in 1.2% glutaraldehyde in stabilization buffer containing 0.2% tannic acid. They then were postfixed in 0.25% OsO4, en bloc stained with 2% aqueous uranyl acetate, and the samples dehydrated in a graded series of ethanols, treated with propylene oxide, and embedded in Epon-Araldite. Ultrathin sections were stained with uranyl acetate and lead citrate, and photographed with a Philips (Eindhoven, The Netherlands) EM400T transmission electron microscope.

52kD ,,,'43kD

,,,,-

Lowicryl-Embedded Immunogold-Labeled Specimens Liver tissue samples were fixed in 4% paraformaldehyde and 0.1% glutaraldehyde with 50 mmol/L lysine in stabilization buffer for 10 minutes at 4°C and transferred to the same fixative without lysine for an additional 50 minutes, followed by an ascending series of ethanols and infiltration overnight at 4°C in Lowicryl K4M. Polymerization was achieved by ultraviolet light at -20°C. Ultrathin sections were mounted on nickel grids. Immunogold labeling was achieved by blocking with 3% bovine serum albumin in phosphate-buffered saline before incubation with the primary antisera for 1 hour. The grids were incubated for I hour with either 10 or 15 nm colloidal gold goat anti-rabbit or mouse gamma G immunoglobulin (Amersham) depending on the primary antibody used, stained with aqueous uranyl acetate and lead citrate, and imaged in a JEOL (JEOL LTD, Akishima, Tokyo, Japan) 1200 EXII transmission electron microscope.

36kD ,,,,-

C

Cryosectioned M i l d l y F i x e d Immunogold-Labeled Specimens Liver tissue samples were fixed in 2% paraformaldehyde for 2 hours. Specimens were infused with 2.3 mol/L sucrose for at least 24 hours, fixed to aluminum microtome pins, and frozen by immersion in liquid N~ and cut in a Reichert Jung (Reichert Jung Optische Werke AG, Wien, Austria) Ultracut E with a FC4 cryo chamber at -90°C on glass knives. Sections were then transferred to carbon formvar-coated nickel grids using molten sucrose, treated with 80 mmol/L ammonium chloride for 10 minutes followed by immersion and quenching in 0.5% bovine serum albumin and 0.15% glycine. Specimens were incubated for 60 minutes with 10 nm colloidal gold goat anti-rabbit or mouse gamma G immunoglobulin (Amersham), depending on the primary antibody. The grids were stabilized with 0.2% uranyl acetate in methyl cellulose before transmission electron microscopy.

FIG. 1. SDS PAGE of bile canalicular membrane homogenate. Distinct bands are detected at 200 kd, myosin II; 130 kd, vinculin; 100 kd, a-actinin; 95 kd, villin; 52 kd, p-tubulin; 43 kd, actin; 52, 45, and 39 kd, cytokeratins; and 36 kd, tropomyosin.

Immunoblotting

t r o p o m y o s i n (36 kd). In SDS P A G E of BC m e m b r a n e isolates blotted on nitrocellulose filters, the antibodies u s e d r e a c t e d w i t h single polypeptide b a n d s at the position of t h e m o l e c u l a r w e i g h t of the respective a n t i g e n s w i t h the exception of the monoclonal a n t i b o d y against cytokeratin. In this instance, t h r e e distinct b a n d s w e r e observed on the nitrocellulose i m m u n o b l o t at t h e app r o p r i a t e m o l e c u l a r weights (corresponding to Moll's peptides #8 [52 kd], #18 [45 kd], a n d #19 [39 kd]) 8 (Fig. 1).

Isolated BC m e m b r a n e p r e p a r a t i o n s c o n t a i n e d polypeptide b a n d s t h a t w e r e specifically labeled w i t h antibodies to m y o s i n II (200 kd), vinculin (130 kd), a-act i n i n (100 kd), villin (95 kd), p - t u b u l i n (52 kd), c y t o k e r a t i n s (52, 45, a n d 39 kd), actin (43 kd), a n d

Electron Microscopy of BC Membranes E l e c t r o n microscopy of n o r m a l h u m a n BC m e m b r a n e p r e p a r a t i o n s p e r m i t t e d g r e a t l y e n h a n c e d precision over tissue sections because of the i n c r e a s e d resolution

RESULTS

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TSUKADA, ACKERLEY, AND PHILLIPS

HEPATOLOGYApril 1995

FIG. 2. (A) Electron microscopy of an oblique section of an isolated bile canaliculus showing the overall architecture of the canaliculusassociated cytoskeleton; in places there are slight disruptions caused by the separation procedure. Large asterisks indicate the canalicular lumen. Three actin microfilament regions are seen. Pairs of small asterisks mark microfilaments associated with the circumferential pericanalicular microfilament band. Membrane-associated microfilaments are marked by small arrowheads, and microfilaments of the microvillous cores are labeled (c). Note also the cross-bridges between the microvillous core filaments and the microvillous membrane (small arrows). Intermediate filaments (large arrowheads), often in bundles, are located predominantly as a sheath around the canaliculus. In the left lower corner of the micrograph, intermediate filaments can be seen inserting into a tangentially sectioned macula adherens plaque (MA). (B) Lowicryl-embedded section of a canaliculus immunogold labeled for actin. Ten-nanometer gold particles are seen surrounding the bile canaliculus (arrowheads), in the microvilli (small arrowheads), and in association with the cell junctional complex (large arrowheads). Zonula occludens (small arrowheads) and the zonula adherens (large arrows) are heavily labeled, but not the macula adherens (MA). obtained. 6 F i g u r e 2A is a t a n g e n t i a l section of a BC t h a t shows m a n y details of t h e m e m b r a n e s t r u c t u r e a n d of t h e u n d e r l y i n g c y t o s k e l e t o n t h a t are difficult or impossible to see in r o u t i n e t i s s u e sections or in t h e i m m u n o - e l e c t r o n microscopic m e t h o d s u s e d on t i s s u e sections. Details of t h r e e m i c r o f i l a m e n t regions can be d i s c e r n e d as well as cross-bridges in t h e microvilli. T h e s u r r o u n d i n g i n t e r m e d i a t e filaments are also well shown.

Immunogold Labeling Lowicryl-Embedded Specimens. E x c e p t for villin, all of t h e antibodies u s e d in this s t u d y labeled specific

c o m p o n e n t s of t h e BC u s i n g L o w i c r y l - e m b e d d e d m a t e rials. I m m u n o g o l d labeling for actin w a s i n t e n s e over t h e c i r c u m f e r e n t i a l m i c r o f i l a m e n t b a n d , t h e microvilli, a n d t h e cell j u n c t i o n s (the z o n u l a occludens a n d t h e z o n u l a a d h e r e n s , b u t not the m a c u l a a d h e r e n s ) (Fig. 2B). M y o s i n I I (Fig. 3A) a n d t r o p o m y o s i n i m m u n o g o l d labeling (Fig. 3B) in t h e s e p r e p a r a t i o n s w a s f o u n d in clusters s u r r o u n d i n g t h e BC in t h e s a m e region as t h e m i c r o f i l a m e n t band. D e n s e labeling w a s observed on t h e z o n u l a a d h e r e n s w i t h a-actinin as well as in clust e r s s u r r o u n d i n g t h e BC (Fig. 3C). S p a r s e gold labeling w a s observed on t h e z o n u l a a d h e r e n s w i t h t h e vinculin a n t i b o d y (Fig. 3E). Most of t h e c y t o k e r a t i n label w a s

HEPATOLOGYVol. 21, No. 4, 1995

FIG. 3. Immunogold labeling of canalicular actin-binding proteins. (A) Cryosectioned, immunogold stained for myosin II. Gold label was observed in merebrahe-associated microfilaments and in the circumferential pericanalicular microfilament band region (arrowheads) and the junctional complex (JC) (*, lumen). (B) Tropomyosin immunogold label. Clusters ofgold particles (arrowheads) are observed in the pericanalicular microfilament regions (*, lumen; JC, junctional complex). (C) Immunogold label for a-actinin. Dense labeling was observed over the zonula adherens (arrows) and in clusters in the pericanalicular microfilaments (arrowheads) (*, lumen; JC, junctional complex). (D) Immunogold label for cytokeratin. The majority of the gold particles were found in the outer aspect of the pericanalicular illament band (arrowheads) (*, lumen). (E) Immunogold label for vinculin. Gold labeling was sparse and confined to the zonula adherens junction (arrowheads) (*, lumen; JC, junctional complex). (F) Immunogold-labeled cryosection that has been stained for villin. Label is confined to BC microvilli (small arrows) (*, lumen). Mag bars = 0.3 ram.

seen in t h e o u t e r portions of t h e p e r i c a n a l i c n l a r illam e n t s a n d the m a c u l a a d h e r e n s ( d e s m o s o m a l ) j u n c tions; occasionally, gold particles were f o u n d in t h e microfilament m e m b r a n e - a s s o c i a t e d region (Fig. 3D). flt u b u l i n i n t e n s e l y labeled t h e m i c r o t u b u l e s seen in the c y t o p l a s m a d j a c e n t to t h e p e r i c a n a l i c u l a r b a n d (not shown). Mildly Fixed Cryosectioned Specimens. The r e s u l t s were s i m i l a r to t h o s e f o u n d w i t h t h e Lowicryl m e t h o d ,

TSUKADA, ACKERLEY, AND PHILLIPS 1109

i ¸ '~!i~!~i~!iii;7 i ~7

i i~i~i~! ~

~i~i

!~!~'I~:;C!

~i~i

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b u t t h e r e were exceptions. Villin antibodies t h a t were n e g a t i v e w i t h Lowicryl labeled i n t e n s e l y the microvilli of t h e BC in cryosections (Fig. 3F). S i n u s o i d a l (basolateral m e m b r a n e ) microvilli f o u n d in the space of Disse did not react. A n o t h e r difference w a s a m a r k e d increase in label for t r o p o m y o s i n a n d m y o s i n I I (Fig. 3A). Tissues k n o w n to c o n t a i n t h e s e proteins a n d in w h i c h t h e i r localizations are well e s t a b l i s h e d (intestinal b r u s h b o r d e r p r e p a r a t i o n s ) were u s e d as controls, a n d

1110 TSUKADA,ACKERLEY, AND PHILLIPS no discrepancies were found (data not shown). A drawing depicting a composite of the cytoskeleton of the bile canaliculus is provided in Fig. 4. It serves to emphasize the general organization of actin microfilaments and of actin-binding proteins; it also demonstrates the relationships of the canalicular cytoskeleton to the components of the junctional complex. It is immediately apparent that these relationships are not random. DISCUSSION

Differentiated epithelial cells are highly specialized in their function, yet there are very close parallels in the general organizational structure of the apical cortex of such cells. 9"~ This basic similarity in structure also extends to ciliated epithelium and is seen throughout phylogeny. 3'~2 One of the best-known examples is the apical region of the intestinal absorptive cell, which serves as a model for such studies. 9'~3-16The fundamental structure, function, and organization of the components of the junctional complex, the zonula occludens, zonula adherens, and the macula adherens, are also well known. 2 The junctional components serve to bind the cells together, are highly ordered, and are always found in the same sequence. They are the attachment sites of distinct cytoskeletal filamentous components of the cell. 2'5'17 One of the characteristics of the apical cytoplasm is a transcellular actin filament belt that traverses the cell and has been shown to be contractile. ~9'~3'15'1s This actin filament belt is attached to the zonula adherens cell junctions. Similar actin filament contractile belts have been described in renal tubule epithelium and in other cell types. 9-1~ In the apical cytoplasm of the hepatocyte, three actin microfilament regions can be observed: BC membraneassociated microfilaments, a circumferential pericanalicular actin microfilament band, and actin associated with microvillous cores. In routine electron micrographs of liver tissue, it is difficult to distinguish them as distinct entities, but in special preparations such as permeabilized liver cells, in canaliculus-enriched liver plasma membrane preparations, and in deep-freeze etch replicas of highly permeabilized rat liver, they are distinct structures. 5'~9 The overall organization is thus similar to that described for other differentiated epithelial cells. In this report, it is shown that each of these actin filament regions has its own complement of actinbinding proteins. The membrane-associated filaments are seen predominantly as a network of microfilaments that attach to the membrane itself; these filaments are associated with myosin II and are likely involved in maintaining the elasticity and structural integrity of the membrane and in the regulation of vesicle transport processes of the canaliculus. The circumferential pericanalicular actin microfilament band (contractile pericanalicular actin filament band) is in the category of one of the more stable microfilament structures that are described in cells 2 and corresponds to the contractile actin filament band seen in the apical cortex of other epithelial cells as cited. ~-11'14'15The filaments in this band are

HEPATOLOGYApril 1995 of mixed polarity, which would enable contractility. 2° Myosin II is known to be located in this region, ~'5'21 and to be involved in contraction movements in nonmuscle cells. 13'22'23 The actin-binding proteins, tropomyosin, aactinin, and myosin II, localize to the pericanalicular actin microfilament band. This is of interest because in muscle cells, a-actinin and tropomyosin are control proteins in the regulation of contraction: for instance, a-actinin is a protein that forms cross-links between actin filaments, providing anchoring points for the actin filaments. 2'11 Both were found in the pericanalicular band in clusters similar to those seen in the terminal web of the intestine and other epithelial cells. 9-11'14'1~ In this study, actin, vinculin, a-actinin, and myosin II are also found in the zonula adherens junctions to which this microfilament band is attached. 911'14 The zonula adherens junction (belt junction) is itself a circumferential membrane plaque situated adjacent to the tight junction (zonula occludens). 2'17'24There is evidence that in some cells, 25'26 possibly including liver c e l l s y actin filaments from the contractile filament band also insert into the tight junction. The actin microfilaments of the microvilli are also in the more stable category and are perpendicularly disposed and arranged in a bundle. Transverse cross-linking of the actin core filaments can also be seen in h u m a n canalicular microvilli, as shown in this report (Fig. 2A). One of the fundamental characteristics of the BC is that any individual liver cell only contributes a hemicanaliculus. Hence, to be a functional unit, there must be a high degree of communication between hepatocytes to coordinate their activity. Evidence for this is suggested from the general morphological observations of the cytoskeleton; both the pericanalicular actin microfilament band and the cytokeratin filament sheath are circumferential (Fig. 2A). It is not known how the two halves of the canaliculus are coordinated, but canalicular contractions are well known to be coordinated and sequential. 2s~° The intermediate filaments need mention in relation to the canaliculus; they form a sheath around the outer perimeter of the canaliculus. 31'32 The function of intermediate filaments in cells is disputed; two functions are proposed here. They serve to stabilize the canaliculus, including the pericanalicular actomyosin contractile band, as suggested also by French et al, 31'32 and they define the boundary of the canalicular compartment of the liver cell. This intermediate filament band inserts into desmosomes (macula adherens junctions). 2 In view of the emphasis placed here on similarities in structure and organization of the apical region of liver cells and other differentiated cells, it must be mentioned that significant differences also exist. One notable difference lies in the microvilli of the BC, which lack the length, rigidity, and constancy of those seen in intestinal absorptive cells; the microvillous actin filament cores are shorter and are also easily lost with effacement of microvilli, as seen in many hepatic disorders. 3s Another difference is in the structure and function of the pericanalicular actin microfilament contrac-

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MACULA ADHERENS cytokeratins, intermediate filaments 2 ZONULA ADHERENS actin vinculin cz actinin 3 ZONULA OCCLUDENS actin 4 MEMBRANE ASSOCIATED MICROFILAMENTS actin myosin II 5 CIRCUMFERENTIAL MtCROFILAMENT BAND actin myosin II tropomyosin cz actinin cytokeratin

6 CYTOKERATIN SHEATH cytokeratin intermediate filaments

7 MICROVILLt actin villin cross bridges ("myosin I") 8 MtCROTUBULES ff tubulin FIG. 4. Drawing of the proposed organization of the cytoskeleton of the bile canaliculus. Three distinct zones of actin microfilaments are depicted: membrane-associated, circumferential pericanalicular microfilament band, and those of the microvillous cores. The localization of actin-binding proteins shown includes their relationship to the components of the junctional complex, which are not random. Note t h a t the actin-binding proteins a-actinin and tropomyosin (contraction control proteins in muscle cells) and myosin 1I are found in the circumferential pericanalicular microfilament band. It represents the BC counterpart of the apical actin filament contractile band seen in other secretory cells. Villin is confined to canalicular microvilli. The cross-bridges joining the microvillous core filaments to the m e m b r a n e are comparable structurally to similar cross-bridges in intestinal microvilli, which have been shown to be myosin I. The intermediate filaments are located predominantly as a s h e a t h t h a t likely stabilizes the bile canaliculus, provides anchoring sites for actin filaments, and defines the outer limits of the bile canalicular compartment.

1112 TSUKADA, ACKERLEY, AND PHILLIPS tile band, w h i c h i n s e r t s into t h e z o n u l a a d h e r e n s (belt) junctions. In t h e liver cell, t h e p e r i c a n a l i c u l a r microf i l a m e n t b a n d can clearly be seen as a distinct f i l a m e n t b a n d only in p e r m e a b i l i z e d cells 19 a n d in isolated bile c a n a l i c u l a r m e m b r a n e s (Fig. 2). It is g e n e r a l l y not as p r o n o u n c e d as it is in o t h e r epithelial cells, p e r h a p s due to t h e c o m p a c t n e s s of t h e apical c y t o p l a s m of t h e liver cell, a n d possibly to a l e s s e r c o n c e n t r a t i o n of t h e a c t o m y o s i n components. A n o t h e r i m p o r t a n t difference in t h e liver is t h a t t h e disposition of t h e p e r i c a n a l i c u l a r actin m i c r o f i l a m e n t b a n d is c i r c u m f e r e n t i a l a r o u n d t h e e n t i r e canaliculus. Hence, c o n t r a c t i o n or r e l a x a t i o n of this b a n d would be e x p e c t e d to p r o d u c e n a r r o w i n g or dilation, respectively, of t h e c a n a l i c u l a r l u m e n , a n d this h a s b e e n r e p o r t e d . 2s'~° A l t h o u g h disputed, 34 t h e evidence for this f r o m in vivo a n d in vitro studies from m a n y l a b o r a t o r i e s is now o v e r w h e l m i n g . 1'21'32'~5-3s F o r example, bile c a n a l i c u l a r c o n t r a c t i o n s can be i n d u c e d by Ca a n d a d e n o s i n e t r i p h o s p h a t e in b o t h p e r m e a b i l ized r a t h e p a t o c y t e couplet cells a n d in isolated h u m a n BC m e m b r a n e p r e p a r a t i o n s , as well as b y t h e microinjection of t h e catalytic c o m p o n e n t of m y o s i n light c h a i n kinase. 1'5'32'37'3sT h e latter, p h o s p h o r y l a t i o n of the regul a t o r y 20-kd m y o s i n light c h a i n b y calcium-calmodul i n - a c t i v a t e d m y o s i n light c h a i n kinase, is responsible for t h e m y o s i n II contractile m o t i o n s e e n in m a n y types of n o n m u s c l e cells. 1,5,13,16,22,23,39O t h e r evidence includes t h e d e m o n s t r a t i o n of r e p e t i t i v e u n i d i r e c t i o n a l contractile m o v e m e n t s in r a t liver in vivo by fluorescent imaging u s i n g t i m e - l a p s e v i d e o - e n h a n c e d microscopy, 3° a n d in a n o t h e r s t u d y in w h i c h co-localization a n d changes in d i s t r i b u t i o n of actin a n d m y o s i n w e r e s h o w n to a c c o m p a n y c a n a l i c u l a r c o n t r a c t i o n in vitro. ~ T h e c a n a l i c u l a r microvilli are u n i q u e in t h a t villin is only f o u n d at this site. Villin is a n actin-binding a n d actin-splicing p r o t e i n ~'~'~9-4~ t h a t binds to t h e actin core filaments of t h e microvillus. Its p r e s e n c e in the i n t e s t i n a l microvilli of t h e chick h a s b e e n well established, 1~'14'4~b u t it is also p r e s e n t in t h e microvilli of t h e bile canaliculus of h u m a n s a n d in m a n y o t h e r a n i m a l species, including the mouse, rat, pig, cow, a n d elep h a n t (personal observations). T h e actin filaments of microvilli are p e r p e n d i c u l a r to the p e r i c a n a l i c u l a r actin belt filaments. T h e r e are rootlets, b u t in h u m a n liver t h e y are v e r y short. It is not c e r t a i n w h e t h e r t h e y i n s e r t into t h e actin f i l a m e n t belt as h a s b e e n clearly s h o w n to be t h e case w i t h core f i l a m e n t rootlets in t h e i n t e s t i n a l b r u s h border. 15 T h e 110-kd p r o t e i n associa t e d w i t h cross-bridges in b r u s h b o r d e r microvilli of t h e i n t e s t i n e was one of t h e first m e m b e r s of t h e m y o s i n 1 s u p e r f a m i l y to be i d e n t i f i e d Y '43 T h e cross-bridges in the microvilli observed in isolated bile c a n a l i c u l a r m e m b r a n e p r e p a r a t i o n s a n d occasionally s e e n in conv e n t i o n a l l y p r e p a r e d t r a n s m i s s i o n electron microg r a p h s of n o r m a l h u m a n liver as i l l u s t r a t e d in this r e p o r t (Fig. 2A) r e s e m b l e t h e 110-kd (myosin I) crossbridges, which are v e r y p r o m i n e n t in i n t e s t i n a l microvilli. T h e f e a t u r e s s h o w n in Fig. 4 serve to s u m m a r i z e t h e g e n e r a l o r g a n i z a t i o n of t h e c a n a l i c u l a r cytoskeleton

HEPATOLOGYApril 1995 w i t h special r e f e r e n c e to actin a n d actin-binding proteins, which are t h e focus of this report. It is clear t h a t not all the f e a t u r e s s h o w n can be seen in a n y single m i c r o g r a p h , b u t e a c h f e a t u r e i l l u s t r a t e d h a s b e e n demo n s t r a t e d . T h e s t r u c t u r a l o r g a n i z a t i o n s h o w n serves to conceptualize t h e a u t h o r s ' i n t e r p r e t a t i o n of the findings. It is likely not the last w o r d on t h e subject b e c a u s e t h e r e is a l r e a d y i n d i r e c t evidence, as cited, t h a t actin f i l a m e n t s from t h e contractile f i l a m e n t belt also e n t e r t h e t i g h t j u n c t i o n (not shown). F u r t h e r , not all cyt o s k e l e t a l s t r u c t u r e s h a v e b e e n depicted; for instance, t h e r e are m a n y m o r e actin-binding proteins, ~ which are not available to the investigators. Moreover, r e s e a r c h on t h e c o m p o n e n t s of the j u n c t i o n a l complex is v e r y r a p i d l y s h e d d i n g new light on t h e s e s t r u c t u r e s . 24'44-46 It was d e e m e d useful, however, to a t t e m p t to s u m m a rize the d i s t r i b u t i o n a n d o r g a n i z a t i o n of k n o w n compon e n t s of t h e canaliculus. REFERENCES

1. Arias IM, Che M, Gatmaitan Z, Leveille C, Nishida N, St. Pierre M. The biologyof the bile canaliculus. HEPATOLOGY1993; 17:318329. 2. Albert B, Bray D, Lewis J, RaffM, Roberts K, Watson JD. Molecular biology of the cell. Ed 2. New York: Garland, 1989. 3. Phillips MJ, Satir P. The cytoskeleton of the hepatocyte: organization, relationship and pathology. In: Arias IM, Jacoby WB, Popper H, Schachter D, Shafritz DA, eds. The liver: biology and pathobiology. Ed 2. New York: Raven Press, 1988. 4. Tsukada N, Ackerley C, Phillips MJ. Organization of the cytoskeleton of the bile canaliculus [Abstract]. HEPATOLOGY 1991; 14:150A. 5. Tsukada N, Phillips MJ. Bile canalicular reorganization of pericanalicular filaments and co-localization of actin and myosin II. J Histochem Cytochem 1993;41:353-363. 6. Laemmli UK. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature (Lend) 1970;227:680685. 7. Towbin H, Staehelin T, Gordon J. Electrophoretic transfer of protein from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc Natl Acad Sci U S A 1979; 76:4350-4354. 8. MollR, Franke WW, Schiller DL. The catalog of human keratins: patterns o£expression in normal epithelia, tumors and cultured cells. Cell 1982;31:11-24. 9. Coudrier E, Kerjaschki D, Louvard D. Cytoskeleton organization and submembranous interactions in intestinal and renal brush borders. Kidney Int 1988;34:309-320. 10. Drenckhahn D, Franke RP. Ultrastructural organization of contractile and cytoskeletal proteins in glomerular podocytes of chicken, rat and man. Lab Invest 1988;59:673-682. 11, Drenckhahn D, Mannherz HG. Distribution of actin and the actin associated proteins myosin, tropomyosin, alpha actinin, and villin in rat and bovine exocrine glands. Eur J Cell Biol 1983; 30:167-178. 12. Reed W, Satir P. Septate junction disruption and the surface reorganization by nonlethal Ca2+ shock. Cell 1979;17:527-535. 13. Broschat KO, Stidwill RP, Burgess DR. Phosphorylation controls brush border motility by regulating myosin structure and association with the cytoskeleton. Cell 1983;35:561-571. 14. Drenckhahn D, Dermietzel R. Organization of the actin filament cytoskeleton in the intestinal brush border: a quantitative and qualitative immune-electron microscope study. J Cell Biol 1988; 107:1037-1048. 15. Hirokawa N, Keller TCS III, Chasa R, Mooseker MS. Mechanism of brush border contractility studied by the quick-freeze, deepetch method. J Cell Biol 1983;96:1325-1336. 16. Keller TCS, Mooseker MS. Cell-calmodulin dependent phosphor-

HEPATOLOGYVol. 21, No. 4, 1995

17. 18. 19.

20. 21. 22.

23. 24. 25. 26. 27. 28. 29. 30. 31.

ylation of myosin and its role in brush border contraction in vitro. J Cell Biol 1982;95:913. Tsukita S, Tsukita S. Isolation of cell to cell adherens junctions from rat liver. J Cell Biol 1989; 108:31. Rodewald R, Newman SB, Karnovsky MJ. Contraction of isolated brush borders from the intestinal epithelium. J Cell Biol 1976; 70:541-554. Watanabe N, Tsukada N, Smith CR, Edwards V, Phillips MJ. Permeabilized hepatocyte couplets: ATP-dependent bile canalicular contractions and a circumferential pericanalicular filament belt demonstrated. Lab Invest 1991;65:203. Ishii M, Washioka H, Tonosaki A, Toyota T. Regional orientation of actin filaments in the pericanalicular cytoplasm of rat hepatocytes. Gastroenterology 1991; 101:1663-1672. Watanabe S, Miyazaki A, Hirose M, Takeuchi M. Myosin in hepatocytes is essential for bile canalicular contraction. Liver 1991;11:185. Lamb NJC, Fernandez A, Conti MA, Adelstein R, Glass DB, Welch WJ, Fermisco J. Regulation of actin microfilament integrity in living nonmuscle cells by the cAMP-dependent protein kinase and the myosin light-chain kinase. J Cell Biol 1988; 106:1955-1971. Kern ED, Hammer JA. Myosins of non-muscle cells. Annu Rev Biophys Chem 1988; 17:23-45. Anderson JM, Balda MS, FanningAS. The structure and regulation of tight junctions. Curr Opin Cell Biol 1993; 5:772-778. Madara JL, Moore R, Carlson S. Alteration of intestinal tight junction structure and permeability by cytoskeleton contraction. Am J Physiol 1987;253:C854-C861. Madara JL. Intestinal absorptive cell tight junctions are linked to cytoskeleton. Am J Physiol 1987;253:C171-C175. Hardison WGM, DaUe-MolleE, Gosnik E, Lowe PJ, Steinback JH, Yamaguchi Y. Function of rat hepatocyte tight junctions: studies with bile acid infusions. Am J Physiol 1991;260:G167-G174. Oshio C, Phillips MJ. Contractility of bile canaliculi, implications for bile flow. Science 1981;212:1041-1042. Phillips MJ, Oshio C, Miyairi M, Katz H, Smith CR. A study of the canalicular contractions in isolated hepatocytes. HEPATOLOaY 1982;2:763-768. Watanabe N, Tsukada N, Smith CR, Phillips MJ. Motility of bile canaliculi in the living animal: implications for bile flow. J Cell Biol 1991; 113:1069-1080. Katsuma Y, Marceua N, Ohta M, French SW. Cytokeratin intermediate filaments of rat hepatocytes: different cytoskeletal domains and their three-dimensional structure. HEPATOLOGY 1988; 8:559-568.

TSUKADA, ACKERLEY, AND PHILLIPS

1113

32. Kawahara H, French SW. Role of cytoskeleton in canalicular contraction in cultured differentiated hepatocytes. Am J Pathol 1990; 136:521-532. 33. Phillips MJ, Poucell S, Patterson J, Valencia P. The liver: an atlas and text of ultrastructural pathology. New York: Raven Press, 1987. 34. Beyer JL. Contractile activity of bile canaliculi: contraction or collapse? HEPATOLOGY1987; 7:190-192. 35. Kitamura T, Brauneis U, Gatmaittan Z, Arias IM. Extracellular ATP, intracellular calcium and canalicular contraction in rat hepatocyte doublets. HEPATOLOGY1991; 14:640-647. 36. Nickola I, Frimmer M. Effects of phalloidin and cytochalasin B on cytoskeletal structures in cultured rat hepatocytes. Cell Tissue Res 1986;245:635-641. 37. Watanabe S, Tomono M, Takeuchi M, Kitamura T, Hirose M, Miyazaki A, Namihisa T. Bile canalicular contraction in the isolated doublet is related to an increase in cytosolic free calcium ion concentration. Liver 1988;8:178-183. 38. Watanabe S, Phillips MJ. Ca ++ causes active contraction of bile canaliculi: direct evidence from microinjection studies. Proc Natl Acad Sci U S A 1984;81:6164-6168. 39. Yamakita Y, Yamashiro S, Matsumura F. In vivo phosphorylation of regulatory light chain of myosin II during mitosis of cultured cells. J Cell Biol 1994; 124:129-138. 40. Craig SW, Powell LD. Regulation of actin polymerization by villin, a 95,000 dalton cytoskeletal component of intestinal brush borders. Cell 1980;22:739-746. 41. Matsudaira PT, Burgess DR. Partial reconstruction of the microvillus core bundle: characterization of villin as a Ca++-dependent, actin bundling/depolymerizing protein. J Cell Biol 1982; 92:648-656. 42. MoosekerMS, ColemanTR. The 110kDprotein-calmodulincomplex of the intestinal microvillus (brush border myosin I) is a mechanoenzyme. J Cell Biol (1989); 108:2395-2400. 43. Cheney RE, Riley MA, Mooseker MS. Phylogenetic analysis of the myosin superfamily. Cell Motil Cytoskel 1993;24:215-223. 44. Furuse M, Hirase T, Itoh M, Nagafuchi A, Yonemura S, Tsukita S, Tsukita S. Occludin: a novel integral membrane protein localizing at tight junctions. J Cell Biol 1993; 123:1777-1788. 45. Gumbiner BM. Breaking through the tight junction barrier. J Cell Biol 1993; 123:1631-1633. 46. Zahraoui A, Joberty G, Arpin M, Fontaine JJ, Hellio R, Tavitian A, Louvard D. A small rab GTPase is distributed in cytoplasmic vesicles in non polarized cells but colocalizes with tight junction marker ZO-1 in polarized epithelial cells. J Cell Biol 1994; 1:101115.