Brain spectrin: A review

Brain Rescorch Ed/din,

0361~923om

Vol. 13, pp. 81L832, 1984. 0 Ankbo International Inc. Printed in the U.S.A.

$3.00 + .oo

Brain Spectrin: A Review STEVEN R. GOODMANt Departments

AND IAN S. ZAGON*

of Physiology? and Anatomy, * The Milton S. Hershey Medical Center The Pennsylvania State University, Hershey, PA 17033 Received 17 December 1984

GOODMAN, S. R. AND I. S. ZAGGN. &oh specrrin: A review. BRAIN RES BULL 13(6) 813-832, 19&t-Red blood cell spectrin, along with actin and several other proteins, forms a skeletal meshwork on the cytopksmic surface of the erythrocyte plasma membrane. This structure is thou&t to maintain red blood ceil shape, membrane structural stability, and celhdar eksticity, as well as controlling the lateral mobility of integral membrane proteins and the tnmsbilayer movement of phospholipids.It is now clearly establishedthat spectrin-relatedmolecules are ubiquitous structural ekmeats subjacent to the plasma membrane of m and avian nonerythroii cells. In this review, we present the current knowledge coacern& brain spectrin. Brain specti is an - 1lS, - I,OOO,OOO molecular weight (a@), tetmmer cont&ning subunits of 240,ooO (Q) and 235,ooO (8) mokcukr weight. It is present in the cortical cytoplasm of ail neuronal cell bodies and processes, and to a ksser extent in glial cells. Its involvement in the actin-membraneinteraction, as well as other proposed functions in the nervous system is discussed. Spectrin Protein 4.1

Brain

Cytoskeleton

Immunocytochemistry

Neurons

Glia

Neurobkstoma

Syndein

Ankyrin

Actin

Neurochemistry

ERYTHROCYTES contain a protein meshwork in close association with the cytoplasmic surface of their plasma membrane. The mar component of this meshwork or membrane skeleton is spectrin, a long fibrous protein which mediates the linkage of actin oligomers Cprotofilaments) to the plasma membmne. In 1981, spectrin (or spectrin-like molecules), long considered to be present only in erythrocytes, was found also to be expressed in a wide variety of nonerythroid cells [33,39]. The discovery of nonerythroid spectrin has generated interest equivalent to that occurring upon the identification of nonmuscle actin and myosin. Nonerythroid spectrin may play major roles in cytoskeleton-membrane interactions, maintenance of the structural stability and elasticity of plasma membranes (and perhaps internal membranes), control of lateral mobility and transbilayer movement of membrane molecules, communication between the cell’s external environment and internal structural elements, and cytoplasmic movement of organelles and membrane vesicles. Based on the intrinsic interest in understanding the cell biology of neural tissue, and the relatively high concentration of spectrin-like protein in brain, brain spectrin (also termed fodrin, calspectin, calmodulin binding protein, and brain actin-binding protein) has received the largest amount of attention of any member of the nonerythroid spectrin family of molecules. It is therefore not surprising that our understanding of tbr structure, protein associations, and cellular localization of this molecule in neural tissue, surpasses our knowledge of nonerythroid spectrin in other tissues. In this review we will: (1) present a brief background on the structure and function of the erythrocyte membrane skeleton, (2) provide an historical account of the discovery of nonerytbroid spectrin, (3) detail our curr?nt understanding of the structure, important interactions, and location of brain spectrin, and (4) discuss potential functions of brain spectrin.

THE ERYTHROCYTESPECTIUNMEMBRANESKELETON

In order to appreciate the basis for searching for nonerythroid spectrin, it is essential to understand the structure and function of the erythrocyte spectrin membrane skekton. A current model of the erythrocyte membrane skeleton is presented in Fig. 1. This is not a static but a dynamic, timeaveraged model in which the skeletal proteins are in an association-dissociation equilibrium with each other and with binding sites on the membrane. The membrane skeletal proteins are continuously reassembling on the cytoplasmic membrane surface in response to both physical and chemical signals during the erythrocyte’s voyage through the circulatory system. The operational deli&ion of the membrane skeleton of erythrocytes has been the proteinaceous reticulum which remains after neutral detergent (e.g., triton X-100) extraction of erythrocytes or erythrocyte membranes (ghosts). The protein composition of the erythrocyte membrane skekton (or Triton shell) is highly dependent on the conditions of extraction, but spectrin is always the major constituent (4075% of the total skeletal protein depending on extraction conditions) [34,75]. In addition to spectrin, the human erythrocyte membrane skeleton contains: actin. syndeins/ ankyrin (bands 2.1 + 2.6). proteins 4. la and 4.lb, band 4.9, and band 7, along with smaller and more variable quantities of band 3, band 4.2 and band 6. A similar composition is found for the membrane skeleton of other species such as pig or mouse, although the components vary in molecular weight, as well as sequence based on pcptide mapping analysis [90]. The molecular interactions of these protein components to form the membrane skekton have been established in considerable detail over the past several years (review (341). Human erythrocyte spectrin (as well as all mammalian

813

xi.4

red blood ceil spectrins) is composed of two distinct [82. 83. 91, 931 Iarge polypeptide chains (bands 1 and 2, or CYand /.!I) (240K and 220K daltons) that form an elongated flexible heterodimer of 1000 A contour length [SO]. The spectrin (o& tetramer, which is formed by head-to-head association of two heterodimers with little or no overlap [80], appears to be the prevalent and physiologically relevant unit of spectrin on the erythrocyte membrane 137, 59, 631; smaller quantities of larger spectrin oligomers aiso appear to be present [60,66]. The spectrin tetramer is bound to the cytoplasmic membrane surface by a high affinity association with a family of peripheral phosphoproteins (bands 2.1, 2.2, 2.3 and ?.h), termed syndeins [91]. Band 2.1 has also been named ankyrin (41. The syndeins are a family of sequence-related proteolytic products of the progenitor polypeptide protein 2. I 1911, which all appear to be present in Go [8I], and function in binding spectrin to the membrane in a stoichiometry of one spectrin tetramer per syndein binding site [37]. Synd~in~Ankyrin bind to the spectrin fl subunit 19,671 approximately 200 A from the junctional site of the heterodimers [87,88]. The syndeins are in turn bound to the membrane by attachment to IO-20% of the t~nsmembrane band 3 molecules [5]. As band 3 (the erythrocyte anion channel) appears to be a tetramer in the membrane and. as there are - I~,~~~ syndeins and 2~~),~~~ the band 3 tetramers per erythrocyte membrane. stoichiometry of the syndein-band 3 interaction would be one syndein bound per every two-to-three band 3 tetramers. Since spectrin tetramers are formed by head-to-head association of heterodimers, other linking proteins are required to form the membrane skeleton network. Short actin ~laments (proto~laments) consisting of --20 actin monomers 1761 and protein 4.1 bind to the terminal ends of the bifunctional spectrin tetramer 187,881, crosslinking adjacent spectrin molecules into a two-dimensional meshwork {reviews 134,361). Human erythrocyte protein 4.1 which appears as a single polypeptide of 80,000 dalton molecular weight on SDS PAGE in continuous buffer systems [22] has been demonstrated to be a family of sequence related peripheral membrane phosphoproteins of 87K, 85K, 8OK, 78K and 67Kdal 1361when analyzed in discontinuous buffer systems [5 lj. The two major members of the protein 4.1 family termed 4. Ia and 4.1 b (80K and 78Kdal) are each present in .- ~00,~ copiesi erythrocyte membrane, and both bind spectrin and stimulate the spectrin-actin interaction 1381. In addition to its involvement in the spect~n-actin interaction, protein 4. I has its own high affinity membrane attachment site distinct from spectrin and actin [78,79], and therefore serves as an additional site of linkage between the spectrin-actin containing skeleton and the cytoplasmic membrane surface. The high affinity attachment site for protein 4.1 has been defined as an integral membrane protein present in --200,000 copies/per erythrocyte 1791, and while several proteins have been purported by others to serve as the protein 4. I binding site, the evidence to date has been contradictory and equivocal (reviews [34,46]). The identity of the binding site for protein 4.1 remains

unresolved.

The spectrin membrane skeleton meshwork is a strong, flexible, and elastic structure, which appears to be essential FACING

for the maintenance of the erythrocyte shape. reversible Jcformability and membrane structural integrity. These functions are essential to the free floating erythrocyte which during its 120-day life span in the circulation must endure continuous Structura] deformation it5 an 8 pin himncaw disc being drawn through capillaries of less than 2 I_L~in diamete) (review [34]). In addition, the membrane skeleton appears to restrict the lateral mobility of integral membrane proteins in the plane of the membrane (review 2311) as weft as being involved in maintenance of the transbilayer phospholipjd asymmetry of the erythrocyte membrane (review 1431). When erythrocyte ghosts are placed in low ionic strength buffers at 37°C. spectrin and actin are specifically removed from the membrane along with small amounts of protein 4. ! : immediately upon dislodging the skeleton, the membrane sheets are completely converted to 1000-3000 n inverted vesicles. This fact, which had been invaluable in studies on cytoskeleto~-membrane interactions, raised the perplexing philosophical question of how plasma membranes of nonerythroid cells which often have lipid compositions and lipid/protein ratio’s which are quite similar to the erythrocyte membrane were able to maintain their structursl integrity. in the absence of an analogue to the spectrin meshwork subjacent to their plasma membrane. This question initiated the search for nonerythroid spectrin in 1980. THE DISCOVERY

OF NONERYTHROID

SPEC-I-RIN

Until 1981, spectrin was believed to be present exclusively in red blood cells. This misconception was based on the fact that although spectrin had been found in all n~ammalian and avian erythrocytes studied, early attempts to demonstrate the presence of spectrin in nonerythroid cells by a complement fixation assay [72] and radioimmunoassay [45] using antibodies against human rbc spectrin were unsuccessful. Yet the philosophical concerns about how nonerythroid cells maintained their membrane structural integrity, and a report of immunoreactive forms of syndei~~nkyrin in diverse cells and tissues [2], suggested that it was necessary to reinvestigate the question of the ubiquity of spectrin. Utilizing several cell types and antibodies to human rbc spectrin, crossreactive proteins were sought in nonerythroid cell types [33,39]. Indirect immunofluorescent staining of embryonic chicken cardiac myocytes (ECCM). mouse fibroblasts (3T3 cells), and rat hepatoma cehs (HTC, HMO,) with monospecific anti-human erythrocytc spectrin IgG revealed a diffuse fluorescence distributed across the entire plasma membrane of permeahitized cells. This fluorescence was not observed when spectr~n-abs~~rbed IgC was substituted in the assay. Furthermore, two spectrin-like polypeptides of 240,000 and 230,000 molecular weight were immunoprecipitated in a 1: 1 (n~ole/mole~ ratio (along with actin, myosin, and other proteins) from octyl glucoside solubilized ECCM with spectrin antibody. Immunoautoradiographic studies demonstrated that among the ECCM immunoprecipitated proteins only the 240,~O and 230,000 dahon peptides stained with anti-erythrocyte spectrin IgG, proving that these peptides represented an immunoreactive analogue of

PAtiE

FIG. 1. Molecular organization of the spectrin membrane skeleton. A model of human erythrocyte membrane skeleton is illustrated. We also present the composition of, and nomenclature for, erythrocyte membrane proteins analyzed by SDS ~lyacrylamid~ gel electropheresis (SDS PAGE) using continuous (left) and discontinuous (right) buffer systems. Drawings presented are equivalent to results obtained when 5% polyacrytamide (continuous system) or 7% polacrylamide (discontinuous system) separating gels are utilized for SDS PAGE. Protein bands on the eels have been color coded to match equivalent proteins on the schematic drawing of the erythocyte membrane skeleton.

815

- SYNDEINS -

I

12.63-ANION TRANSrjoRT CHANNEL w 3_

4.2-6 \ -7

4.5ACTIN

-6

\

4.9, 5-

cGLCX3IN 6-

GOODMAN TABLE CELLS AND TISSUES

AN]) %AOON

1

IN WHICH SPECTRIN-RELATED MOLECULES HAVE BEEN LOCALIZED

Chronological

References

Tissue Bladder Brain (neurons and glial) Gall Bladder Small and Large Intestine Kidney Lens (cortical cells) Liver (hepatocytes) Lymphocytes Cardiac Muscle Embryonic Cardiac Myocytes Skeletal Muscle Uterus

~231 (56, 26, 74, 23, 40, 41, 11. 921 ]231 156, 26, 74, 46, 30, 231 ~231

[74, 55, 23. 701 ]231

[57,681 ]231 [391 [56, 26, 74, 70, 23, 141 ~231

Cell Cultures Bovine Adrenal Medulla Chromaffin Cells Mouse Adrenal Tumor Cells (Y 1)

WI Osawa et ul. (manuscript submitted)

Human Amnion Epithelial Cells Marbin-Darby Canine Kidney (MDBK) Epithelial Cells Potaroo Kidney (PTK) Epithelial Cells Bovine Embryonic Fibroblasts Chick Embryonic Fibroblasts Human Embryonic Fibroblasts Mouse 3T3 Fibroblasts Mouse C3H lOT% Fibroblasts Gerbil Fibroma Cells C,; Glioma Cells HeLa Cells Rat Hepatoma Cells (HMDA) Monocyte Derived Macrophages Human Neuroblastoma Cells Mouse Neuroblastoma Cells (S2OY) Superior Cervical Ganglion Neurons

]551 ]55,621

Sertoli Cells

erythrocyte spectrin. This unique method of immunoprecipitation followed by immunoautoradiography proved to be an extremely sensitive technique for detecting minor immunoreactive analogues in cells, and has found wide applicability in the nonerythroid spectrin field. To determine the structural relationship between ECCM spectrin and embryonic chicken rbc spectrin, one-dimensional proteolytic peptide mapping on the individual subunits of both proteins was performed. The 240,000 dalton subunit of ECCM spectrin shared substantial peptide homology with the embryonic chicken rbc 240,000 dalton (Ysubunit, while the 230,000 dalton subunit of ECCM spectrin shared substantial peptide homology with the p subunit of embryonic chicken rbc spectrin. These studies demonstrated, for the first time, that immunoreactive and structural analogues of rbc spectrin exist in diverse noneryfhroid cells [33,39]. As in many pivotal studies, there was a good deal of serendipity in the choice of embryonic chicken cardiac myocytes as the first tissue to receive extensive biochemical and immunological investiga-

]551

[621 ]26,231 ]551 [33, 56, 391

PI P.621 [561 [26.621 i33.391 ]551 ]551 Goodman and Zagon (presented

here)

1561 [61

tion, because in avian tissues all spectrins contain a highly related u subunit 123.241, and cardiac muscle is one of the few nonerythroid tissues which contains a /3 subunit which is nearly identical to erythrocyte /3 spectrin [70]. Had another cell type been chosen with a spectrin-like molecule which was further removed in structure from erythrocyte spectrin. the discovery of nonerythroid spectrin might have been delayed. Demonstration of the presence of a spectrin-like protein in nonerythroid cells has led to the search for spectrinrelated molecules in diverse cells and tissues. In the last three years it has become clear from immunofluorescence and immunoelectronmicroscopy studies carried out in many laboratories using antibodies against rbc spectrin, brain spectrin, and terminal web (TW) 26Oi240 that spectrin-like molecules can be found on or near the plasma membrane in the cortical cytoplasm of every avian and mammalian cell or tissue which has been studied. A list of the cells and tissues already demonstrated to contain nonerythroid spectrin is presented in

BRAIN SPECTRIN

817

Table 1. Elegant studies by Glenney and Glenney [231 have demonstrated that in avian tissues three different major spectrin forms are found. Red blood cells and skeletal muscle contain spectrin molecules which have highly related 240,000 dalton (Ysubunits as well as related 220,000 or 230,000 dalton p subunits. Most other cells and tissues including brain, lymphocytes, fibroblasts, liver, kidney, bladder, gall bladder, and lens contain a spectrin molecule which consists of a 240,008 dalton LYsubunit and a 235,000 dalton p subunit (sometimes referred to as fodrin). Chicken intestine contains a unique protein which has a 240,000 dalton a subunit and a 260,000 dalton fi subunit (TW260/240), in addition to the widely found spectrin (240/235). Heart contains both the 2401235 spectrin as well as the 2401220 or 2401230 rbc type spectrin. It is important to note that these studies have been performed with avian tissues; and whether a similar distribution will be found in mammalian tissues is unclear. At this time it appears that the distribution in mammalian tissues may not be identical as mammalian intestine does not appear to contain TW2601240 [25]. Two nonerythroid spectrins have received a great deal of attention (TW260/240 and brain spectrin). TW260/240, isolated from the brush borders of chicken intestinal epithelial cells, is an elongated double stranded molecule of 2600 A contour length that binds to and crosslinks actin filaments by attachment at both of its ends [26]. The protein contains a 240,008 dalton a! subunit which is structurally and immunologically related to chicken rbc spectrin a subunit, as well as sharing a binding site for calmodulin [28]. TW2601240 arises later in embryonic development than intestinal spectrin (240/235) which is found on both the apical and basolateral membranes of intestinal epithelial cells [23]. TW260/240 is located in the brush border crosslinking actin bundles to actin bundles, as well as attaching actin filaments to intermediate filaments, actin to the membrane, actin to coated vesicles, and coated vesicles to membranes [30,46]. The structure, location, and function of brain spectrin has also been studied in great detail, and these topics will be discussed later. BRAIN

Early

SPECTRIN

Studies

Concurrent with attempts to identify nonerythroid spectrin [33,39], several laboratories were studying the functional aspects of a high molecular weight actin and calmodulin binding protein in brain, unaware that they were characterizing a spectrin-related molecule. In 1981, Davies and Klee purified a high molecular weight doublet containing polypeptides of 235,000 and 230,000 daltons from bovine brain on the basis of its ability to bind calmodulin [ 151. While this calmodulin binding protein also could bind f-actin, these authors were unable to demonstrate immunological crossreactivity with antibodies against human rbc spectrin, and the protein was termed calmodulin binding protein-I (CBP-I). Simultaneously, Kakiuchi and coworkers isolated a 240,000 dalton peptide from a 6 M urea extract of bovine brain on a calmodulin affinity column [47]. As this protein did not appear to contain the characteristic spectrin doublet, these authors did not suspect that they were analyzing the spectrin (Y subunit, and no immunologic studies were attempted. Shimo-Oka and Watanabe isolated a protein from pig brain with subunits of 240,000 and 235,000 daltons which could bind to actin, and stimulate actomyosin Mg+*-ATPase [77]. The smaller subunit did not comigrate with rbc spectrin /3

subunit, and no immunological data was available, and these authors named this protein brain actin-binding protein (BABP). Levine and Willard in an elegant characterization of two axonally transported polypeptides of 250,000 and 240,000 daltons, raised antibodies against electrophoretically prepared guinea pig protein, and demonstrated by immunofluorescence that this protein doublet was highly concentrated at the internal periphery of neurons, Schwann cells and a wide variety of non-neuronal cells and tissues [56]. While this axonally transported protein bound actin, attempts to demonstrate a reaction between antibodies against this protein and guinea pig rbc spectrin by Ouchterlony analysis were negative. It was therefore concluded that this was not a spectrin-related protein, and it was given the name fodrin (from Greek fodros=lining) because of its ring-like presence in the cortical cytoplasm of neural and non-neural cells. All of these early studies shared the common property that, while they yielded essential information on the actin and calmodulin binding properties and location of this protein in brain, they lacked recognition of the relationship of this brain protein to spectrin; this led to the multiple assignments of trivial names. A similar situation occurred with the myosin family of molecules, where initially new names were coined for the equivalent nonmuscle proteins, but subsequent work demonstrated sufficient structural and functional homologies to justify calling them all myosins. The demonstration of nonerythroid spectrin-like molecules in diverse cells [33,39] led several laboratories in the following year to reinvestigate the relationship between these high molecular weight actin and calmodulin binding proteins in brain and red blood cell spectrin. Antibodies against the purified brain protein (240Kdal and 235Kdal subunits) cross-reacted with red blood cell spectrin [8, 28, 401 and antibodies against red blood cell spectrin were found to crossreact with brain spectrin [3,40,73] as well as MAP-2 [16]. Furthermore, Palfrey and colleagues were able to immunoprecipitate the calmodulin binding protein from avian brain with antibodies against avian rbc (Y spectrin [73]. In Fig. 2, antibodies against mouse rbc spectrin (lanes A and B) can be seen to crossreact with the spectrin-like protein from mous. brain (lanes C and D), staining both the 240,000 and 235,000 subunits (lanes G-J). Antibodies against mouse brain spectrin-like protein (lanes E-F) stain almost exclusively the 240,000 dalton subunit (lanes K-N) [ 11, 40, 41, 921. It therefore became clear that the protein from brain is an immunoreactive analogue of rbc spectrin. Furthermore, as discussed in subsequent sections, several laboratories also demonstrated that red blood cell spectrin and brain spectrin share several important morphological and functional properties [3, 8, 26281. Kakiuchi et al. reinvestigated their calmodulin binding protein from bovine brain, and found that when isolated under milder conditions this calmodulin and actin binding protein had subunits of 240,000 and 235,000 daltons [48].Given the prevailing evidence that this protein was another member of the nonerythroid spectrin family of molecules, they gave still another appellation to this protein, and called it calspectin [48]. As it is clear that (a) all of these proteins CBP-I, brain actin binding protein (BABP), fodrin, and calspectin are all the same, (b) they are structurally, functionally, and immunologically related to red blood cell spectrin, and (c) that all of the nonerythroid spectrin-like proteins probably evolved from a common ancestoral gene, the term brain spectrin might be more reasonable. Thus all of the nonerythroid spectrin-like proteins can simply be named by the tissue or cell in which they are found, followed by the

A

G D

E F

GHI

J

KLMN

FIG. 2. Mammalian erythrocyte and brain spectrin are immunoreactive analogues. SDS PAGE and immunoautoradiography by the gel overlay method was performed as described [22,491. On lanes G-N electrophoresis was extended for 4 hours, to allow clear separation of the 240,000 and 235,000 dalton subunits of brain spectrin. ‘The gels containing: mouse erythrocyte membrane protein (A), mouse brain membrane protein (C,E.C,K) and pure mouse brain spectrin (H,L) were incubated with anti-mouse erythrocyte spectrin l[gG (B,D,I,Y) or anti-mouse brain spectrin IgG (F,M,N) followed by i*“Y] Protein A. The autoradiographs were exposed for 1 day (B,F,M,N), 6 days (D) or 10 days (I,J) at -20°C. Molecular weights are given as M.W. x 10-“.

B

2

BRAIN sPIXTRI~

A

6

CDECOHIJ

KLMNOPQRS

FIG. 3. Isolation of mouse brain spectrin. SDS PAGE was performed as described [22]. The coomassie blue stained gel contains human erythrocyte membranes (A). demyekted braio membranes (B), demyelinated braia membraaes depleted of b&n spectrin by low ionic strength extraction (C), low ioaic strength extract of crude brain spectrin (D), and 15 fractions obtain upon rate xonal sedimentation of low ionic strength extract thru a S-l&%sucrose gradiit (J&S). E is the bottom of the gradient and G-J contain p&tied brain spectria. Taken from Goodman et al. [41] with the pubfisher’s permission.

term spectrin (e.g., brain spectrin, liver spectrin, HeLa spectrin). In tissues in which more than one spectrin analogue exists, the nomenchunre should include the moiecular weight of the subunits (x lO-3} in parentheses, e.g., chicken brain spectrin(2W235) and chicken brain s~ct~(~~~. Utilixation of this nomenclature should avoid much of the confusion being generated by the current multitude of names found in the literature. BRAINSPECTRiNSTRW’MJRE Brain spectrin contains two polypeptide chains in equal molar ratios of 240,ooO and 235,i.W molecular weight (see Fii. 2 lane II), which compose 24% of the total protein in crude brain membrane preparations [3, 8, 26, 40, 561. This protein has been extracted at high ionic strength [26,56] and low ionic strength 13, 8, 401 from brain membranes, and purified by gel filtration with a huge pore gel [3,8,26,56] or by rate xonal sedimentation through a sucrose gradient [40]. As shown in Fig. 3, in our laboratory we prefer the low ionic strength extraction which is more selective and less prone to proteolytic degradation of brain spectrin. Using this method

50% of the spectrin is extracted from the mouse brain membraues (lanes ED), and brain spectrin can be isolated in high yield and purity by the one simple step of rate xonal sedimentation (Ianes G-J contains pure brain spectrin). The typical yield of mouse brain spectrin is -0.5 mg per 20 g of mouse brain [40,41]. The mouse brain spectrin has a calculated molecular weight based on its hydrodynamic properties (S,,= 10.5, Stokes Radius=220 A, partial specific volume of 0.730) of 972,000 d&tons. This molecular weight is very close to the estimate of 950,ooO caiculated for an (cY#?~ tetramer with subunits of 240,090 (a) and 235,080 (rs)daltons. Proteins of similar structure and subunit composition have been isolated from chicken, pig, and cow brain membranes [3,8,26J. Therefore brain spectrin isolated from ah of these species is a 1-e asymmetric tetramer with two copies of each subunit(a&. These properties of brain spectrin are nearly identical to the red blood cell spectrm molecule which is an llS, -l,OOO,alO moMllar weight (a& t&ramer (review [34]). Rotary shadowed electron microg@rs of brain spectrin demonstrate a long flexible rod of m A contour length, with the two strands woven into a tight double helix with few gaps, which is tightly associated at both ends 13, 17, 26,271.

820

The morphology of brain spectrin (illustrated in Fig. 4) is nearly identical to the erythrocyte spectrin tetramer which is a slightly looser double helix with a contour length of 2000 A [80]. Parenthetically, chicken intestinal spectrin(260/240) also resembles the double stranded morphology of rbc spectrin although it has a longer contour length of 2600 A [26]. Apparently ail members of the nonerythroid spectrin family share this elongated, double stranded, helical structure. The fact that the brain spectrin molecule is formed by head-tohead interaction of two 2401235 heterodimers, rather than being a linkage of a 240-240 homodimer to a 235-235 homodimer, was most clearly demonstrated by the antibody mapping technique of Glenney et al. [29]. Monoclonal antibodies against specific epitopes on the cy and p subunits of brain spectrin were observed on rotary shadowed micrographs to decorate two points on a brain spectrin molecule which were equal distances from the two ends. This bilateral symmetry of monoclonal antibody binding to brain spectrin tetramers [29] is consistent with the bilateral symmetry of actin, syndein/ankyrin, and calmodulin binding sites [3, 27, 861, and leads to the conclusion that the brain spectrin tetramer must be formed by head-to-head interactions to two cwp heterodimers (see Fig. 4). When two monoclonal antibodies were bound to a brain spectrin molecule in which the strands between the antigenic sites could be traced, it was clear that the antibodies were situated on opposite strands [29], therefore the head of a 240,000 dalton (Ysubunit must be contiguous with the head of a 235,000 dalton /3 subunit to form one morphologically continuous strand (the second strand is built the same way but with opposite polarity). This model for brain spectrin demonstrated in Fig. 4, is identical to the current model for erythrocyte spectrin in which the 240,000 dalton (Ysubunit of one heterodimer is paired with the 220,000 dalton /3 chain of an adjacent heterodimer and vice versa [80]. Unlike the erythrocyte spectrin tetramer, the brain spectrin tetramer is not easily disassociated into individual heterodimers [3]. However, in the presence of high urea concentrations, the brain spectrin tetramers disassociate into individual a: and /3 subunits; upon removal of the urea these individual subunits can reassociate into heterodimers [ 171. Interestingly hybrid erythrocyte-brain heterodimers can be formed when the cy subunits of brain spectrin are combined with the /3 subunits of erythrocyte spectrin (or vice versa), indicating that both molecules must share common terminal association sites [ 171. Peptide mapping analysis of brain spectrin and erythrocyte spectrin have revealed some extremely important distinctions between avian and mammalian spectrin. Chymotryptic peptide mapping of avian brain and rbc spectrin 240,000 dalton (Ysubunits have yielded identical peptide patterns, while the 235,000 dalton brain subunit shows a distinct map from the 220,000 dahon rbc spectrin p subunit [23-25, 281. This suggests that the 240,000 dalton (Y subunit of chicken rbc and brain spectrin may be coded for by a single gene, while the 235,000 and 220,000 dalton /3 subunits of brain and rbc spectrin are clearly distinct gene products. The mammalian spectrins differ significantly from the avian spectrins. Chymotryptic peptide maps of pig brain and rbc spectrin [3, 24, 251 and tryptic peptide maps of mouse brain and rbc spectrin [40,41], alI show that little peptide homology exists between either the 240,000 dalton LYsubunits or the 235,000 and 220,000 dalton /3 subunits of mammalian brain and erythrocyte spectrin (see Fig. 5). In summary, while avian spectrins have a constant OLsubunit and a variable /3 subunit, mammalian brain spectrin has both a variable (Yand

GOODMAN

AND ZAGGN

FIG. 4. Model of the brain spectrin molecule. The morphology of the 2000 A long brain spectrin molecule is presented, along with the bivalent binding sites for calmodulin (Cal). syndein/ankytin (Syn/Ank), and actin. Computer drawing by Dr. Carol Whitfield.

/3 subunit when compared to mammalian rbc spectrin. The distinctive peptide maps observed for mammalian brain and rbc spectrin has led to the hypothesis that the mammalian brain and rbc spectrins probably have relatively small highly conserved functional regions of sequence such as the syndei?/ankyrin and actin binding sites, which are separated by long domains of variable sequence (functionally “silent domains”) [34, 40, 411. The extended rod-like conformation of spectrin would be expected to be more compatible with the variation of amino acid sequence proposed for these silent domains, than the folding of a globular protein. Furthermore, peptide mapping analysis on mammalian spectrins suggests that both the (Yand /3 subunits of brain and rbc spectrin are distinct gene products, a point which is supported by the finding that sphlsph mice. which cannot synthesize red blood cell spectrin leading to a severe spherocytic hemolytic anemia, produce normal levels of brain spectrin 240,ooO and 235,000 da&on subunits [I I]. These differences between avian and mammalian spectrins show that extrapolation of findings between spectrin in avian brain to mammalian brain must be made cautiously. In addition to the points raised previously, chicken red blood cells contain a variant of @ spectrin with a molecular weight of 230,000 daltons (p’) (in addition to the 220Kdal subunit) [70]; this variant p subunit is not found in any mammalian rbc spectrin. Lazarides and Nelson [52,53] have reported that in addition to brain spectrin(240/235), chicken brain contains a minor form of brain spectrin(240/230 or 240/220) which is highly related to red blood cell spectrin. This minor form of chicken brain spectrin(240/230 or 2401220) has not yet been detected in mammalian brain. Because of the two types of spectrin in chicken brain, Lazarides has suggested that the 235,ooO dalton subunit, which is structurally distinct from chicken rbc /3 spectrin be called y spectrin [52.53]. Since the 235,000 dalton subunit of brain spectrin has a brain syndein/ankyrin binding site [18,19], and association sites for the (Ysubunit of rbc as well as brain spectrin [17], it fulfills the functional requirements of a spectrin p subunit and we will refer to it as such in this text. In addition to the morphological similarities between brain and erythrocyte spectrin which have been described above, and the functional similarities which will be described in subsequent sections, brain and rbc spectrin are both phosphorylated in their /I subunit [4l]. In viva and in rifro studies by Goodman and colleagues has demonstrated that the 235,000 da&on p subunit of mouse brain spectrin and the 220,000 dalton /3 subunit of mouse rbc spectrin are both phosphorylated by a membrane-associated CAMP independent protein kinase 1411. The human rbc spectrin molecule is phosphorylated by a CAMP independent protein kinase at 4 sites within 10,000 daltons of the c-terminal end of the /j

BRAIN SPECTBIN

821

CHROMATOGRAPHY FIG. 5. Twodimensional tryptic peptide mapping analysis of mouse erythrocyte and brain spectrin. Two-dimensional tryptic peptide analysis of proteins radioiodinated within the gel slice was performed by the method of Elder et al. [21J. Subunits (a and /3) of erythrocyte spectrin and brain spectrin were clearly separated by extending SDS PAGE on a low percentage acrylamide gel for 4 hr. Tryptic peptide analysis of erythrocyte spectrin a subunit (A), erythrocyte- spectrin /3 subunit (B), brain spectrin a subunit (C), and brain spectrin /3 (D) are demonstrated. Note the lack of homologY between the brain and erythmcyte spectrin subunits. Taken from Goodman er al. [41] with the publisher’s permission.

chain, close to the junctional hcterodimer association sites WI. TIN number of phosphates, the position of phosphates, and the function of the phosphate groups on mouse brain and mouse rbc spectrin remains to be determined. BRAIN SPECTRIN-MEMBRANE INTERACTIONS(ROLEOF BRAIN SYNDEINANKYRIN) How is brain spectrin associated with the plasma membrane (and internal membranes) of neural cells? While we do not yet know the answer to this question, we have some interesting leads based on the identification and isolation of a

syndein/ankyrin mokcule from the plasma membrane of neural cells [ 18,191.As previously described, even before the identilication of nonerythroid spectrin [33,391, Bennett had aheady demonstrated the presence of immunoreactive syndein/ankyrin analogues in diverse cells and tissues [21. Brain spectrin was shown to bind to the syndein!ankyrin bindin site on erythrocyte spectrindepleted vesicles, demonstrating that brain spectrin shared with rbc specttin a common binding site for red blood cell syndein/ankyrin [3,8]. But does brain contain a membrane-associated sy&inIankyrin analogue which can serve as a spectrin binding ptotein? Mammalian brain membranes contain polypeptides of 22OKdal,

GOODMAN

x22

210Kdal and 15OKdal (similar to rbc syndein/ankyrin) which crossreact with rbc ankyrin antibodies [18]. The membraneassociated brain syndein/ankyrin could be cleaved with chymotrypsin into a 72,000 dalton soluble fragment and a 95,000 dalton membrane-associated fragment [181, resembling the spectrin and band 3 binding domains of red blood ceil syndeimankyrin [89]. The 72,ooO dalton fragment of brain syndeinlankyrin was capable of binding rbc spectrin. Recently brain syndein/ankyrin has been substantially purified (80% purity) from pig brain membranes [19]. The brain syndeimankyrin has a calculated molecular weight of 178,000 daltons based on its hydrodynamic properties (SZ,,,w=6.2, Stokes radius=68 A, partial specific volume=0.73). The purified brain syndeinlankyrin bound to brain .spectrin in solution with a K,,=25 nM and a stoichiometry of 2 syndein/ankyrin molecules per brain spectrin tetramer. Rotary shadowed micrographs of this interaction demonstrated that the binding sites for brain syndeinlankyrin on brain spectrin reside 800 A from each end of the tetramer (see Fig. 4). Blotting analysis indicated that brain syndeinlankyrin binds to the 235,000 dalton p subunit of brain spectrin [19]. The affmity of the interaction, the location of the binding site, and the association with the 235,000 dalton p subunit of brain spectrin, are remarkably similar to the rbc spectrinsyndein/ankyrin interaction 1341. The 95,000 dalton membrane-associated domain of brain syndein/ankyrin can bind to the cytoplasm& domain of rbc band 3 [19]. Does brain contain a Band 3 analogue? Antibodies against human rbc band 3 have been demonstrated to crossreact with peptides of 69,000 and 60,000 daltons in mouse neuroblastoma cells [50] and other nonerythroid cells [20,50]. Whether these peptides contain a binding site for brain syndeinlankyrin and are present as an integral component of neural plasma membranes is unknown. However, the studies described above present the possibility that brain spectrin may be associated with the plasma membrane of neural cells at least in part through a spectrin-syndein/ankyrin-band 3 interaction. Of course binding of brain spectrin to the cytoplasmic surface of well characterized spectrin-depleted neural plasma membrane preparations must be conducted, before it can be concluded that brain syndeinlankyrin functions in situ in the attachment of brain spectrin to membranes. BRAIN

SPECTRIN-ACTIN

INTERACTION

Several laboratories have presented evidence that brain spectrin can bind to and crosslink actin filaments, by sedimentation and low shear viscometry measurements [3,8, 26, 27, 56, 771. Glenney and coworkers [27] have demonstrated by rotary shadowing of the brain spectrin-f-actin complex, that brain spectrin binds end-on to actin filaments. This is the identical image observed on rotary shadowing of the rbc spectrin tetramer-f-actin interaction (review [34]). As the brain spectrin tetramer is bivalent (having binding sites at both of its termini) it can crosslink adjacent actin filaments (see Fig. 4). The existence of a fibrous meshwork subjacent to the plasma membrane of many nonerythroid cells including nerve cells has long been recognized by electron microscopists, yet the composition of these subcortical skeletons are only beginning to be understood. It is quite likely that the long (2000 A), slender (-40 A width) brain spectrin molecule may play a key role in mediating the association of cortical actin to the plasma membrane of neural cells, as well as linking the cortical actin filaments to each other forming a three-dimensional fibrous mat subjacent to

AND ZAGON

the membrane. Recent evidence also suggests that nonerythroid spectrin may be involved in the association of intermediate filaments with the plasma membrane [62], and in the association of actin filaments with intermediate filaments [46]; furthermore, brain ankyrin has been suggested as a potential binding site for tubulin on the plasma membrane [ 191. Therefore spectrin and syndeinlankyrin in brain may be key structural components of a three-dimensional membraneassociated matrix involving cortical actin filaments, intermediate f%unents, and microtubules. BRAIN 4. I

The red blood cell spectrin-f-actin interaction is extremely weak in solution being converted to a high affinity interaction in the presence of protein 4.1 through formation of a ternary complex (Fig. 1). Two hemolytic anemias, hereditary spherocytosis (HS) and hereditary elliptocytosis (HE) are, in some instances, due to a defective HS spectrin4.1~,actin interaction [35] or absence of protein 4.1 (HE) [85]. Therefore protein 4.1 is essential in maintaining the shape and structural integrity of red blood cell membranes. Two laboratories have demonstrated that erythrocyte protein 4.1 can strengthen the interaction of brain spectrin and actin filaments in vitro [7,58], suggesting that brain spectrin contains a protein 4.1 binding site. But does brain have a protein 4.1 analogue? Goodman and colleagues have used an antibody against native erythrocyte protein 4.1 to demonstrate that pig brain has an 87,000 dalton immunoreactive analogue of protein 4.1 (Fig. 6), which colocates with spectrin in the cortical cytoplasm of neuronal cell bodies and processes throughout the cerebellum [32]. The immunoprecipitated pig brain protein 4.1 shared approximately 50% peptide homology with pig rbc protein 4.1, on two dimensional tryptic peptide analysis (Fig. 7). This study [32] represented the first demonstration of brain protein 4.1. Studies to determine whether brain 4.1 can stimulate the brain spectrin-actin interaction and/or serve as a membraneattachment site for brain spectrin, are currently underway. It appears that all of the major components of the red blood cell membrane skeleton (spectrin, syndein/ankyrin, protein 4.1, and of course actin) are present in brain, raising the intriguing possibility that a subcoritcal skeleton exists in brain with a related composition and function to the red blood cell membrane skeleton. Granger and Lazarides have recently reported that protein 4.1 is expressed only in avian red blood cells and lens cells, but not in avian cerebellum, sciatic nerve, small intestine, chicken embryo fibroblasts, skeletal muscle or any other cell or tissues [42]. It is not yet clear whether this negative result reflects another difference between avian and mammalian systems, or alternatively demonstrates the inherent difficulties in using antibodies against denatured protein 4.1 for detecting native globular protein 4.1 (with only partial sequence homology to rbc protein 4.1) by indirect immunofluorescence. CA”

DEPENDENT

CALMODULIN

BINDING

All avian spectrins, including brain spectrin, have a 240,000 dalton LYsubunit which can bind calmodulin in the presence of Ca++; mammalian brain spectrin (Ysubunit also binds calmodulin, but mammalian rbc spectrin either binds weakly or not at all when the binding is studied by a gel overlay method [ 10, 15, 28,47. 731. The location of the bind-

BRAIN SPECTBIN

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2. #

3 4.

43 l

a

b

a

bed

FIG. 6. Demonstration of an 87.000 dalton analogue to rbc 4.1 in pig brain. (A) Pig rbc membraaes and protein 4.1 isolated as described [38] were subjected to SDS PAGE [22]. Coomassie blue stained gels contain pig rbc membrane protein (lane a) and puritiid pig rbc protein 4.1 (lane b). (B) Immunoautoradiography by the gel overlay method was performed as described [49]. (lane a) Coomassie blue stained pia rbc membrane protein. (lane b) Matching autoradiogram of proteins in hme a which stain with pig rbc 4.1 IgG. (lane c) Coomassie blue stained preparation of crude pig brain membranes. (lane d) Matching autoradiogram of proteins in lane c which stain with the pig rbc 4.1 IgG. Autoradiography was performed at -20°C for 1 day (lane b) or 5 days (lane d). Taken from Goodman et al. [32] with the publisher’s permission.

ing site for calmodulin on the brain spectrin molecule has been determined by rotary shadowing and electron microscopy of the brain spectrin-calmodulin complex [8f5], Calmodulin binds towards the center of the brain spectrin tetramer at a distance of about 75 A from the junction of the heterodimers (Fig. 4). The function of the cahnodulin-brain spectrin interaction is not known, although preliminary experiments suggest that calmodulin does not affect the brain spectrin-actin interaction [ 151. It will be interesting to determine whether calmodulin regulates the phosphorylation of brain spectrin. IMMUNOCYTOCHEMICAL LOCALIZATIONOF SPECTRININ NEURAL TISSUE

To understand the function of brain spectrin, structural and functional studies described above must be coupled with

careful localization of spectrin with specific neuronal and glial cell types. The locahxakn of spectrin in the nervous system has been reported by a number of laboratories. Levine and Willard [56], using an antibody to the a subunit of brain spectrin examined the cervical spinal cord, optic tract, medial longitudimd fascicuks, dorsal root ganglion neurons, spinal motor neurons, and sciatic nerve of guinea p@s, as well as rat superior cervical ganglion cell cultures, rat retinal explant cultures, and C-6 glioma cells in tissue culture. These workers recorded a conceutration of antigen in the cork&¶ cytoplasm of neurons, as well as large dendrites and the axon initial segments. In cross-sections of the sciatic nerve, a bright rhq of fluorescence comsponding to the periphery of the axou was recorded. Upon bigher magnification, Levine and Wii discovered an unstained annulus, 2-3 pm in width surrounding the intensely stained

FIG. 7. Tryptic peptide analysis of pig erythrocyte and brain protein 4.1. Affinity-purified IgG to pig rbc protein 4.1 (28 /.q$ was used to immunoprecipitate protein 4.1 from pig rbc and brain membranes solubilized in Triton X- 100 at high ionic strength [23]. Immunoprecipitated pig brain and rbc &otein 4.1, as well as total pig brain and rbc membrane proteins were separated by SDS PAGE [22]. Two-dimensional tryptic peptide mapping qnalysis of peptides labeled with lz51within the gel slice ga$ performed [21]. (A) Tryptic map of pig rbc protein 4.1 excised from a gel containing total ghost protein. (B) Tryptic map of immunoprecipitated pig rbc protein 4.1. (C) Tryptic map of immunoprecipitated pig brain protein 4.1. Some of the common tryptic peptides are indicated (arrows). Taken from Goodman et cl/. 1321with the publisher’? permission.

axolemmal region; beyond this annulus, a faintly stained ring was also noted. In longitudinal sections, the nodes of Ranvier were clear@ resolved. These investigators interpreted the unstained annulus as the region of the myelin sheath surrounding the ;axon, whereas the stained outer ring corresponded to the plasma membrane of the Schwann cell. Electron microscopic analysis will be required to define whether spectrin is in the axon and the outer plasma membrane of Schwann cells, or is in the inner and outer mesaxons of the Schwann cell where glial cytoplasm is concentrated. A number of other reports also have briefly described the localization of spectrin in neural tissues and cells, corroborating Levine and Willard’s observation as to spectrin’s concentration in the cortical cytoplasm. These include (a) Repasky et al.‘s [74] report showing staining of chicken sciatic nerve and’ spinal cord with antibodies to chicken erythrocyte (Yspectrin, (b) Glenney et af.‘s [26] demonstration of staining of mouse nerve bundles in tongue and sciatic nerve with antibodies to chicken TW260/240, (c) Glenney and Glenney’s, ,[23] study with antibodies to chicken rbc 235K subunit using chicken sciatic nerve, and (d) Lehto and Virtanen’s [55] report of (Y spectrin ant&ens in human neuroblastoma cells in culture. The localization of chicken brain spectrin(240/235), and brain spectrin(240/230 or 220) which is closely related to rbc spectrin in neural tissues of chicken has been examined by Lazarides and colleagues [52, 53, 541. In a brief report, Lazarides and Nelson [52] showed that the brain spectrin (2401235) is in virtually all neural cell bodies and processes of 7-week old chickens, whereas the erythrocyte form was confined to the plasmalemma of neuronal cell bodies. Based on their observations, these authors sugge’sted that there is a mechanism for segregating different spectrin complexes into distinct membrane domains within a single cell. In subsequent studies, Lazarides and co-workers examined the distribution of brain and erythrocyte forms of spectrin in the developing chicken cerebellum [53] and optic system [54]. The brain form of chicken spectrin was localized in all neuronal cell bodies and processes during all stages of cere-

bellar morphogenesis. However, the erythrocyte form of spectrin was detected in the plasma membrane of the cell bodies of Purkinje, granule, and deep cerebellar neurons only after these cells became postmitotic and completed their migration.The appearance of the erythrocyte form of spectrin coincides temporally with the establishment of axosomatic contacts on these 3 neuronal cell types, and the authors suggested that the erythrocyte form accumulates in response to the formation of functional synaptic connections during ontogeny. Utilizing the chicken optic system, Lazarides, Nelson and Kasamatsu [54] have demonstrated that the brain-specific form of spectrin is a major membrane-associated, axonally transported cytoskeletal protein in adult brain. This form of spectrin is present in all developing layers of the retina at all stages of embryogenesis examined, with the ganglion cell layer and the developing axons of the optic nerve showing particularly intense fluorescence. The erythrocyte form of spectrin was present at the plasma membrane of only the ganglion cell bodies and a subset of the processes in the inner plexiform layer close to the ganglion cell layer which are presumed to be the dendritic processes of ganglion cells and that extend into the inner plexiform layer of the avian retina. The axonal processes of optic nerve fibers within the retina also stained with antibodies to the erythrocyte form of spectrin, an observation that differs from the absence of axonal staining in the chicken cerebellum reported by Lazarides and Nelson [53]. Lazarides and colleagues also noted that the cell bodies and processes of amacrine, bipolar, and horizontal cells present in the inner nuclear layer, the outer plexiform layer, and the bacillary layer were unstained except for the cell bodies of the rods and cones in the outer nuclear layer. Developmentally, the onset of the erythrocyte-form of spectrin appears to coincide with the phase of synaptogenesis. These results with the chicken cerebellum and optic system suggest the existence of a developmentally regulated mechanism that topologically segregates the erythroid and brain forms of chicken spectrin from each other, and the former from axonal transport. Lazarides and co-workers have suggested

FIG. 9. Spectrin locaiizatian in tt anti-rbc spectrin. In contrast to a re~auve~~ un~wneu nuc~ar rogwz~11 processes (arrows) ~930 (Al; ~660 (B).

BRAIN SPECTIUN

827

1O.spCetrinlocdiatioainesarobhstormcells.Mtvinene~~~(S#Iy),48brincultun,~~laou#~~ spectrin (A) er mouse anti-rbc specbkt (It). The cortical cytoplasm and cell process (arrows)exhibit intense -seeace, but cell nuclei (n) showed littk immmmrestivity. Growth cones (cross-hatched arrow) and dividing ceils (Te=tebphase) were extremely intense. x455.

FIG.

that erythroid spectrin may be involved in establishing restricted membrane-cytoskeletal domains in neurons during synaptogenesis, and maintaining them in the adult cell. Although spectrin-like mokcules appear to be ubiquitous in all animal tissues, it may not be appropriate to equate the distribution and assembly of these molecules across avian and mammalian species. As discussed in detail earlier, while all mammalian rbc spectrins contain a 240Kdal ar subunit in association with a 220Kdal /3 subunit [34,90], chicken rbc spectrin contains a 22OKdal/3 subunit and a 230Kdal /I’ subunit individually associated with a 240Kdal ar subunit [70]. In addition while all nonerythroid spectrins from chicken contain an a subunit which is identical by peptide mapping analysis and a variable ~3subunit, the mammmalian spectrins demonstrate variable a and jl subunits (as described above). Thus far no evidence for a minor red blood cell form of spectrin containing a 230Kdalfl’ subunit in association with an OLsubunit has been demonstrated in mammalian brain. In a series of studies, Goodman and Zagon [11,40,41,921 have begun to explore the distribution of spectrin in the mammalian nervous system. In utilizing antibodies to mouse brain spectrin or etythrocyte spectrin, immunofluorescent studies revealed an intense staining of the cortical cytoplasm of internal granule neurons in the mouse (Fig. 8A and B) and pig cerebellum, as well as granule cells of the dentate gyrus in mouse. In a systematic study of the distribution of spectrin in the central nervous system of mammals [92], utilixing sag&al and coronal sections of the mouse brain and an erythrocyte spectrin antibody which crossreacted specifically

with the a and /3 subunits of brain spectrin, a fuller understanding of spectrin’s location was obtained. Spectrin antigens were concentrated in the cell bodies of all neurons, and were recorded even in the finest cell processes (Fig. 8A-C, Fig. 9A and B). Synaptic structures and axons were observed to have little detectable spectrin analogue by immunofluorescent methodology. The basis for the limited staining of axons and synaptic structures in the mammalian central nervous system with antibodies against erythrocyte spectrin is not at present clear, but is being pursued by immunoelectronmicroscopy. The medullary layer of cerebellum, the corpus callosum, mammillo&uamic tract, fomix, internal capsule, corticospinal tract, anterior commisure, and cerebellar peduncles exhibited little staining. Nerve fibers preserved in these preparations, as argyrophilic ele-. merits, were visualixed by Rotargol staining of preparations which were previously examined with fluorescence microscopy * GM cells had a less intense immunoreactivity in their cell bodies, but neither glial cell nuclei nor processes were stained. The choroid plexus had low to moderate immunoreactivity, but the cortical cytoplasm of ependymal cells was found to be distinctly fluorescent. In the ventricular region, prominently stained fibrous elements were often observed, but their origin was undetermined. In unpublished observations, Goodman and Zagon have found that murine S2OY neuroblastoma cells in culture exhibit dramatic immunofbtorescent staining with antibodies to mouse rbc spectrin or mouse brain spectrin (Fig. 1OAand B).

X28

GOODMAN

Even the finest cell processes were visible, and the cytoplasm of dividing cells was extremely fluorescent. The nuclei of neuroblastoma cells were unstained. NeurobIastoma ceils express a spectrin molecule with subunits of 240,000 and 235,000 daltons (Fig. 11). This cell line should prove valuable in studies on the assembly of the spectrin based membrane skeleton. Based on a complete study of spectrin localization in mammalian brain, a number of basic principles can be defined: (1) Spectrin is found in all regions of rna~~an brain and its intensity corresponds to the density of neural cells. (2) Different neural cell types contain variable spectrin content; for example, neurons contain more immunoreactive spectrin than do glial cell types. (3) Within a single cell type the regional disposition of spectrin varies; for example, neurons display a brightly stained cytoplasm of the cell body and dendrites, little staining of axons, and no nuclear staining. Of course, these i~uno~uorescent studies do not supply a quantitative view of relative spectrin content in different neural cells. For example, the less intense staining of neuronal axons or glial cell types could be due to a spectrin molecule that has fewer antigenic sites which can be recognized by a particular spectrin IgG, as compared to the spectrin molecule found in the neuronal cell bodies. Moreover, to obtain a specific intercellular localization for spectrin in neural cells, electron immunomicroscopy is mandatory. It is important to note that some studies on other cytoskeletal proteins closely associated with spectrin have been published. Protein 4.1 is a membrane skeletal protein that stimulates the weak, end-on, bivalent binding of spectrin tetramer to F-actin. Imm~ocytochemic~ investi~tions [32] reveal that brain protein 4.1 colocalizes with spectrin, being prominent in the neuronal cortical cytoplasm and processes but not in the nucleus (Fig. 12). While protein 4.1 appears to be colocated with spectrin throughout brain tissue at least at the level of the light microscope, protein 4.1 in Ebroblasts has been reported to be associated with stress fibers suggesting a different unction for 4. I in those cells [ 131.

POTENTIAL FUNCTIONS OF BRAIN SPECTRIN

When thinking of the potential functions of spectrin in brain, it is appealing to begin with those properties which are analogous to the erythrocyte spectrin membr~e skeleton. Therefore spectrin may be involved in (a) membrane anchorage of cortical actin, (b) control of the shape of neural cells, (c) maintenance of neural plasma membrane structural integrity (and internal memb~es?) (d) regulation of the lateral mobility of integral membrane proteins (e.g., receptors), (e) maintenance of plasma membrane phospholipid asymmetry and (0 allowing synaptic terminals their plasticity. However, as we have discussed in previous sections the functions of brain spectrin are probably more complex than red blood cell spectrin, and may include linking intermediate filaments to membr~es, intermediate ~laments to actin filaments, actin fdaments to actin filaments, and perhaps (thru syndei~~ky~n} associating microtubules to membranes. It therefore appears that brain spectrin may be a central component in a three-dimensional meshwork located just beneath the plasma membrane of neural cells. As suggested by the quick-freeze deep etch electron micrographs of the spectrin-related molecules in intestinal epithelial cells 1461, spectrin may also function in nonerythrnid cells (including neural cells) in the attachment of coated and

AND ZAGON

1 2

A

BCD

FIG. 11, Expression of neuroblastoma spectrin(24W.35). Mouse neuroblastoma cells (S2OY)preincubated with S:‘“-methionine were sotubilited in triton X-100 at high ionic strength. Speotrin was immunoprecip~~ted with monospecific anti-mouse brain spectrin IgG as described [23]. (Lane A) Erythrocyte membrane proteins as standards. (Lane B) Autoradiograph of the total S”s-iabelled neuroblastoma proteins. (Lane C) Auto~diograph of immunopreci~itated Ss5-labefled neuroblastoma spectrin(2401235). (Lane D) Autoradiograph of control in which preimmune IgG was substituted in the incubation for anti-brain spectrin IgG. Note the clean immunoprecipitation of newly synthesized Reurobiastoma spect~n~240/23S).

smooth vesicles to actin filaments, and vesicles to plasma membr~e. Therefore spectrin may be involved in the intracellular traEic of membrane vesicles. The neural spectrin cortical skeleton is undoubtedly a dynamic structure, and it has been suggested by Willard and colleagues that brain spectrin(240/235) may be part of a mobile lining in the cortical cytoplasm of the cell bodies and axons of neurons, which is essential for the movement of organelles, vesicles, and cytoskeletal com~nents to their correct location with the cell [ 121. It is also possible that spectrin mediates changes in the cytoskeleton in response to external stimuli such as neu-

829

BRAIN SPECTRIN

FIG. 12.~4.l~~~.(A)A~ npictureof&sl@talsuctiunofthumousuALES top41:pprotein4.1.&ights~oftheiatc~~~~yer~GL)andlowtomoderabestainiqsdtbt~ (NED) layers is showa. x 312,(R and C) Granuleneurons iu the lGL of the mouse (B) aud pig (C) are immunoreactiveto pig rbc proteiu4.1. Note the brightly fluorescent cortical cytoplasm (ruvows)encirclinga noufIuorusciugnucleus. x860. ~~s~~r attachment to cell surface receptors, or contacts between extracellular matrix components and the neuronal plasma membrane. Spectrin may be involved in synaptic function, possibly in Ca+2 induced release of transmitter substances at the nerve terminals [al]. while we can now only guess at the functions of spectrin in brain, the tremendous amonnt of current research interest in the fun&ion of these molecules may well, witbin the next few years, convert some of our conjectures into revelations.

AC~OWLE~E~S

This work was suppurkd ht part by NationalInsthutes of Health Grants NS-19357sad HL-26059to S. R. Goodmau and NS-21246to I. S. Zagou. S. R. Goodman is aa Established Rkvestigatorof the American Heart &so&ion. We that& Patrick hfaese and huumtte Schwartz for the+ diligent worh in prc@ng the tnaeu&pt, and David Sitteraad PatriciaJ. McLaughgnfor B sss~cc. As~~~~~~S.~~~~e~

picture of the membraae skeletoa prestated in Fig. 1.

I. Appleyard, S. R., M. J. Dunn, V. Dubowitz, M. L. Scott, S. J. Pittman and D. M. Shotton. Monoclonal antibodies detect a spectrin-like protein in normal and dystrophic human skeletal muscle. Prtjc Nati Acud Sci USA 81: 776780, 1984. 2. Bennett, V. Immunoreactive forms of human erythrocyte ankyrin are present in diverse cells and tissues. Nazctw 281: 597-599. 1979. 3. Bennett, V., J. Davis and W. E. Fowler. Brain specttin. a membrane-associated protein related in structure and function to erythrocyte spectrin. Nuture 299: 126131, 1982. 4. Bennett. V. and P. J. Stenbuck. Identification and vmartial auriti--cation of ankyrin, the high afBnity membrane attachment site for human erythrocyte specttin. J Biol Chem 254: 2533-2541. 1979. 5 Bennett, V. and P. J. Stenbuck. The membrane attachment protein for spectrin is associated with band 3 in human erythrocyte membranes. Nature Zso: 468-473, 1979. 6 Borland, K., S. Osawa, D. Kew, D. B. Coleman, S. R. Goodman and P. F. Hall. Identification of a spectrin-like protein in Sertoli cells. J Reprod Viol, in press. 7. Burns, N. R., V. Ohanian and W. B. Gratzer. Properties of brain spectrin (fodrin). FEBS Iett 153: 165-168, 1983. 8. But-ridge, K., T. Kelly and P. Mangeat. Nonerythrocyte spectrins: Actin-membrane attachment proteins occurring in many cell types. J CPN Biol 95: 478-486, 1982. 9. Calve& R., P. Bennett and W. B. Gratzer. Properties and structural role of the subunits of human spectrin. EuurJ Biothem 107: 355-361, 1980. 10. Carlin, R. K., D. C. Bartelt and P. Siekevitz. Identification of fodrin as a major calmodulin-binding protein in postsynaptic density preparations. J Ceil Biol 96~ 443-448, 1983. Il. Casotia, L. A.. I. S. Zagon, S. Bernstein, S. Shohet. P. J. McLaughlin and S. R. Goodman. Normal content of brain spectrin-like protein in SphtSph mice. Br J Huemarol 58: 659667, 1984. 12. Cheney, R., N. Hirokawa, J. Levine and M. Willard. Intracellular movement of fodrin. Cell Notih?y 3: 649-655, 1983. 13. Cohen, C. M., S. F. Foley and C. Korsgren. A protein immunologically related to erythrocyte band 4.1 is found on stress libres of non-erythroid cells. Nature 299: 648-650, 1982. 14 Craig S. W. and J. V. Pardo. Gamma actin, spectrin, and intermediate fdament proteins colocalize with vinculin at costameres, myofib~l-t~s~colemma attachment sites. (‘cl/ ~(~rjlif? 3: 449462, 1983. 15. Davies, P. J. A. and C. B. Klee. Calmodulin-binding proteins: A high molecular weight calmodulin-binding protein from bovine brain. Biochcm Inr 3: 203-212, 1981. 16. Davis, J. Q. and V. Bennett. Microtubule-associated protein 2. a microtubule-associated protein from brain, is immunologically related to the a subunit of erythrocyte spectrin. J Aiol (‘hem 257: 58165820, 1982. 17. Davis, J. Q. and V. Bennett. Brain spectrin: Isolation of subunits and formation of hybrids with erythrocyte spectrin sub units. J Biol Chum 258: 7757-7766. 1983. 18. Davis, J. Q. and V. Bennett. Brain ankytin: purification of a 72,ooO M, spectrin-binding domain. J Biol C‘hem 259: t87C 1881. 1984. 19. Davis, J. Q. and V. Bennett. Brain ankyrin: A membraneassociated protein with binding sites for spectrin. tubulin, and the cytoplasmic domain of the erythrocyte anion channel. J Biol (‘hem 259: 13550-13559, 1984. 20. Drenckhahn, D.. K. Zinke, U. Schauer, K. C. Appell and P. S. Low. Identification of immunoreactive forms of human erythrocyte band 3 in nonerythroid cells. Elrr I (‘(,!I Biol 34: 144-150. 1984. 21. Elder, 1. H., R. A. Pickett. II, J. Hampton and R. A. Lemer. Radioiodination of proteins in single polyacrylamide gel slices. J Biol Chrm 252: 6510-6515. 1977. 22. Fairbanks, Cl.. T. L.. Steck and D. F. H. Wallach. Elcctrophoretic analysis of the major polypeptides of the human erythrocyte membrane. Biochemist~f IO: 2606-2617, 1971. I

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23. Glenney, J. R., Jr. and P. Glenney. Fodrin is the general spectrin-like protein found in most cells whereas spectrin and the TW protein have a restricted distribution. C’~,ll34: 503-5 13. 1983. 24. Glenney, J. R., Jr. and P. Glenney. Spectrin. fodrin, and TW260/2& a familv of related proteins lining the plasma membrane. (‘eli Motility 3: 67 1-682,. 1983. 25. Glenney, J. R.. Jr. and P. Glenney. Comparison of spectrin isolated from erythrocyte and non-erythroid sources. /+u .I Bbchmr. in press. 26. Glenney, J. R.. Jr., P. Glenney. M. Osborn and K. Weber. An F-actin- and c~m~ulin-bindin protein from isolated intestinal brush borders has a morphology related to spectrin. Cefi 2H: 843-854, 1982. 27. Glenney, J. R., Jr., P. Glenney and K. Weber. F-actin-binding and cross-linking properties of porcine brain fodrin, a spectrinrelated molecule. J Biol Chum 257: 9781-9887, 1982. 28. Glenney, J. R., Jr., P.-Glenney-and K.. Weber. Erytbroid spectrin, brain fodrin, and intestinal brush border proteins (TW.260/240) are related molecules containing a common c~m~ulin-binding subunit bound to a variant cell type-specific subunit. f’roc Narl Acad Sci USA 79: 4002-4005, 1982. 29. Glenney, J. R., Jr.. P. Glenney and K. Weber. Mapping the fodrin molecule with monoclonal antibodies. .I &!(#I Viol 167: 275-293, 1983. 30. Glenney, J. R., Jr., P. Glenney and K. Weber. The spectrinrelated molecule, TW-260t240, cross-links the actin bundles of !he microvillas rootlets in the brush borders of intestinal epithclial cells. J CPN Biol 96: 1491-1496. 1983. 3 1. Goodman, S. R. and D. Branton. Spectrin binding and the control of membrane protein mobility. J .Supr~o~~~~/ .S~rucI 8: 455,463. 1978. 32. Goodman, S. R., L. A. Casoria, 1). B. Colemanand 1. S. Zagon. identification and location of brain protein 4.1. Stkvu~c~ 2% 1433-1436, 1984. 33. Goodman, S. R. and R. R. Kulikowski. Identification of spectrin in nonerythroid cells. .I Srrpromol Strut C’C+ Rio&vt (Suppl) 5: 261. 1981. 34. Goodman, S. R. and K. A. Shiffer. The spectrin membrane skeleton of normal and abnormal human erythrocytes: a review. Am J Ph.v.sio/ 244: 121-141. 1983. 35. Goodman, S. R., K. A. Shiffer, L. A. Casoriaand M. E. Eyster. Identi~cation of the molecular defect in the erythrocyte membrane skeleton of some kindreds with hereditary spherocytosis. Btwd 60: 772-784, 1982. 36. Goodman, S. R., K. S. Shiffer. D. B. Coleman and C. F. Whitfield. Erythrocyte membrane skeletal protein 4.1: A brief review. In: The Red Cell: Sixth Ann Arbor ~‘wjkenc~. edited by G. Brewer. New York: AIan R. Liss. 1984, pp. 415-439. 37. Goodman, S. R. and S. A. Weidner. Binding of spectrin n& tetramers to human erythrocyte membranes. .I &J/ (‘hem 255: 80828086, 1980. 38. Goodman, S. R., J. Yu, C. F. Whitfield. E. N. Culp and E. J. Posnak. Erythrocyte membrane skeletal protein bands 4. la and b are sequence-related phosphoproteins. J Hi<)/ f‘h~wz 257: 4564-4569. 1982. 39. Goodman. S. R.. I. S. Zagon and R. R. Kulikowski. Identification of a spectrin-like protein in nonerythroid cells. Prtjc, Nrrrl Acud So’ USA 78: 7570-7574. 1981. 40. Goodman, S. R.. I. S. Zagon, C. F. Whitfield, L. A. Casoria. P. J. McLaughlin and T. L. Laskiewicz. A spectrin-like protein from mouse brain membranes: Immunological and structural correlations with erythrocyte spectrin. (‘~11 Motilirv 3: 635-647. 1983. 41. Goodman, S. R., I. S. Zagon, C. F. Whit~eld, L. A. tasoria. S. B. Shohet. S. Bernstein, P. J. McLau8hlin and T. L. Laskiewicz. A spectrin-like protein from mouse brain membranes: Phosphorylation of the 235,ooO dalton subunit. Am .I Physiol 16: c’6H.73. 1984.

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%AC;ON

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