The fodrin-ankyrin cytoskeleton of choroid plexus preferentially colocalizes with apical Na+K(+)-ATPase rather than with basolateral anion exchanger AE2. S L Alper, … , D Brown, D Drenckhahn J Clin Invest. 1994;93(4):1430-1438. https://doi.org/10.1172/JCI117120. Research Article A unique feature of the choroid plexus as a single-layer epithelium is its localization of Na+K(+)-ATPase at its apical (lumenal) surface. In contrast, a band 3 (AE1)-related anion exchanger protein has been localized to the basolateral surface of the choroid plexus. Both Na+K(+)-ATPase and AE1 in other tissues have been shown to bind via ankyrin to the spectrin-actin-based membrane cytoskeleton. Since linkage of integral membrane proteins to the membrane cytoskeleton is important for their restriction to specialized domains of the cell surface, we investigated the polarity of the choroid plexus membrane cytoskeleton. We developed isoform-specific antibodies to confirm the identity of choroid plexus band 3-related polypeptide as AE2. We demonstrated that ankyrin, fodrin/spectrin, actin, myosin, and alpha-actinin are predominantly apical in choroid plexus and preferentially colocalize with apical Na+K(+)-ATPase rather than with basolateral anion exchanger AE2. Colchicine administration did not alter the polarity of apical cytoskeletal and transport proteins or basolateral AE2 in choroid plexus, suggesting that biosynthetic targeting of these proteins is not microtubule dependent. In choroid plexus papilloma, Na+K(+)-ATPase and AE2 were decreased in amount and failed to preserve their polarized distributions. Find the latest version: https://jci.me/117120/pdf The Fodrin-Ankyrin Cytoskeleton of Choroid Plexus Preferentially Colocalizes with Apical Na+K+-ATPase Rather than with Basolateral Anion Exchanger AE2 Seth L. Alper,*11 Alan Stuart-Tilley,* Charles F. Simmons, Dennis Brown,Ol and Detlev Drenckhahn** *Molecular Medicine and Renal Units, Beth Israel Hospital; tDivision ofNeonatology, The Children's Hospital; Renal Unit, Massachusetts General Hospital; IlDepartments of Cell Biology and 'Pathology, Harvard Medical School, Boston, Massachusetts 02215; and **The Anatomical Institute, University of Wilrzburg, 97070 W~rzburg, Germany Abstract brane proteins, such consensus sequences have not been found for plasmalemmal proteins (1). A unique feature of the choroid plexus as a single-layer epithe- In addition to targeting signals within protein sequences, lium is its localization of Na+K+-ATPase at its apical (lu- other targeting mechanisms have been investigated. These in- menal) surface. In contrast, a band 3 (AE1 )-related anion ex- clude heterotrimeric (2) and low molecular weight G proteins changer protein has been localized to the basolateral surface of (3) as vesicle targeting and docking agents and selective reten- the choroid plexus. Both Na+K+-ATPase and AEI in other tion and stabilization by association via ankyrin to the spec- tissues have been shown to bind via ankyrin to the spectrin-ac- trin/actin-based membrane cytoskeleton (4, 5). The latter tin-based membrane cytoskeleton. Since linkage of integral mechanism, in particular, has been invoked to explain the po- membrane proteins to the membrane cytoskeleton is important larized steady state distribution ofanion exchanger 1 (AE 1 ) ' in for their restriction to specialized domains of the cell surface, the basolateral membrane of renal collecting duct intercalated we investigated the polarity of the choroid plexus membrane cells (5) and of the Na+K+-ATPase in the basolateral mem- cytoskeleton. We developed isoform-specific antibodies to con- brane of Madin-Darby canine kidney (MDCK) cells (4) and firm the identity of choroid plexus band 3-related polypeptide kidney tubules (6, 7). One clonal line of MDCK cells appears as AE2. We demonstrated that ankyrin, fodrin/spectrin, actin, to deliver nascent Na+K+-ATPase randomly to basolateral myosin, and a-actinin are predominantly apical in choroid and apical cell surfaces but selectively stabilizes the protein at plexus and preferentially colocalize with apical Na+K+-ATP- the basolateral surface, apparently through binding to ankyrin ase rather than with basolateral anion exchanger AE2. Colchi- (4,8). cine administration did not alter the polarity of apical cytoskele- In most polarized epithelial cells, immunoreactive ankyrin tal and transport proteins or basolateral AE2 in choroid plexus, is concentrated at the basolateral membrane (5, 9). However, suggesting that biosynthetic targeting of these proteins is not several cell types express ankyrin-associated P-type ATPases in microtubule dependent. In choroid plexus papilloma, the apical membrane. For example, the apicovesicular localiza- Na+K+-ATPase and AE2 were decreased in amount and failed tion of the H+ K+-ATPase in rabbit gastric gland parietal cells to preserve their polarized distributions. (J. Clin. Invest. 1994. appears to be accompanied by apical localization ofimmunore- 93:1430-1438.) Key words: choroid plexus - epithelial polarity active ankyrin (10). Another cell type which expresses apical * anion exchanger 2 . sodium-potassium adenosine triphospha- ankyrin is the retinal pigment epithelium, in which tase * cytoskeleton Na+ K+-ATPase colocalizes to the apical surface ( 1 1). How- ever, this double-layer epithelium is unique in that its apical rather is in N-CAM-stabilized con- Introduction surface is not lumenal but tact with photoreceptor cells ( 12). The choroid plexus, in con- with a lumenal to trast, is a simple, single-layer epithelium apical The great majority of plasma membrane proteins examined surface. It is the only such epithelium which expresses apical date in polarized epithelia display polarity of expression. The Na+K+-ATPase. Interestingly, the choroid plexus also secretes mechanisms ofplasma membrane protein targeting remain un- apically into the cerebrospinal fluid (CSF) transthyretin, ceru- known. Initial experiments focused on the search for targeting loplasmin, and cystatin C, all proteins which are secreted baso- signals within the primary sequences of the polarized mem- laterally by hepatocytes ( 13-15). The cDNAs which encode brane proteins. Though operationally defined targeting signals these secreted proteins have all been cloned from both choroid have indeed been found in a small number of single-span plexus and from liver and have been found not to differ in the to date for multi- membrane proteins, none has been defined two cell types ( 16). In addition, peptide map, immunochemi- ple-span membrane transport proteins. Whereas consensus and 19) have indicated that for mem- cal ( 17, 18), transcript analyses ( targeting sequences have been identified organellar choroid plexus expresses the a 1 subunit of the Na+K+-ATP- ase, as well as ,BI and 32 subunits. The unusual apical expres- Portions ofthis work have appeared in abstract form ( 1991. Kidney Int. sion and secretion ofthese polypeptides, biochemically indistin- 38:71 la). guishable from their basolateral counterparts in other epithelial Address correspondence to Dr. Seth L. Alper, Molecular Medicine has to the that the choroid has MA 02215. tissues, led hypothesis plexus Unit, Beth Israel Hospital, 330 Brookline Avenue, Boston, a "reversal" ofelements of its mech- Received for publication 12 August 1993 and in revised form 25 undergone protein-sorting October 1993. anism. In addition to the Na+K+-ATPase, another membrane J. Clin. Invest. transport protein expressed in abundance in choroid plexus is © The American Society for Clinical Investigation, Inc. 0021-9738/94/04/1430/9 $2.00 1. Abbreviations used in this paper: AEl, anion exchanger 1; CSF, Volume 93, April 1994, 1430-1438 cerebrospinal fluid; MDCK cells, Madin-Darby canine kidney cells. 1430 Alper, Stuart-Tilley, Simmons, Brown, and Drenckhahn the band 3-related chloride/bicarbonate exchanger AE2 (20, placed directly into SDS load buffer, or fixed in 3% paraformaldehyde, 21). AE2 shares with the erythroid anion exchanger (AE1, quenched, and suspended in 3% agar-PBS which was allowed to solid- band 3) conserved sequences within their cytoplasmic do- ify, and fixed for embedding (37). Transgenic mice were monitored for mains which have been proposed as binding sites for nonery- the development of ataxia and/or cerebellar bossing. Mice showing throid homologs of erythroid cytoskeletal proteins, including these physical signs were anesthetized, and choroid papilloma tumor and surrounding choroid plexus were dissected as above. ankyrin (22, 23). The choroid plexus presents a situation in Fertilized chick eggs at embryonic day 13 were gifts of Dr. D. Wu which a known ankyrin-binding protein, Na+K+-ATPase, is and Dr. C. Cepko. Choroid plexus was fresh-dissected into artificial localized apically (24, 25) while a proposed ankyrin-binding CSF and handled as above. Pig choroid plexus was obtained by dissec- protein, AE2, is localized basolaterally (21 ). tion ofheads obtained after surgery from the Tufts University School of In this paper we characterize isoform-specific antibodies to Veterinary Medicine (Medford, MA). AE2 and use them to confirm AE2 as the band 3-related pro- Transfections. p7OZN8 (22) encoding murine AE2 protein and tein of the basolateral membrane of the choroid plexus. We pL2AA encoding murine kidney AE 1 (38) were transiently transfected localize AE2 and the Na+K+-ATPase to opposite surfaces of into COS-1 cells using the DEAE-dextran procedure (38, 39). [35S]- the choroid plexus epithelium. We show that immunoreactive Methionine