Proc. Nat. Acad. Sci. USAVol. 69, No. 2, pp. 471-474, February 1972

Membrane Microfilaments of Erythrocytes: Alteration in Intact CellsReproduces the Hereditary Spherocytosis Syndrome

(vinblastine/colchicine/strychnine/electron microscopy/cell rigidity)

HARRY JACOB, THOMAS AMSDEN, AND JAMES WHITE

Departments of Medicine and Pediatrics, University of Minnesota Medical School, Minneapolis, Minn. 55455

Communicated by Robert A. Good, November fB, 1971

ABSTRACT Membrane microfilaments are foundthroughout the animal world in situations suggestingthat they fulfill a critical role in providing normal cellshape and plasticity. We have hypothesized that hereditaryspherocytosis, a congenital hemolytic anemia associatedwith intrinsically rigid and mishapen erythrocytes, mightresult from genetically defective microfilaments in eryth-rocyte membranes. By using three different drugs(vinblastine, colchicine, and strychnine) that share onecommon attribute-that of potently precipitating purifiedmicrofilamentous protein-we have provided support forthis hypothesis. Thus, all the known in vitro and in vivocharacteristics of hereditary spherocytes are reproducedin normal erythrocytes briefly exposed to these precipitat-ing agents.

Hereditary spherocytosis, the most prevalent congenitalhemolytic anemia of man, affects roughly 0.02% of all hu-mans. Evidence has accumulated that an intrinsic defect in theerythrocyte membrane underlies this disease. This evidenceincludes: (a) erythrocyte membranes in this disease leak so-dium at excessive rates (1, 2); (b) "buds" of membrane formprematurely, and fragment off the hereditary spherocyteduring incubation, thereby enhancing spherocytosis (3,4); (c)erythrocyte ghosts from affected individuals are intrinsicallystiffer than normal, resisting passage into glass microcapillarytubes (5); and (d) these membranes have a unique "wrinkled"appearance when they are viewed with surface-scanningelectron microscopy (6). The nature of the membrane defecthas recently been studied through examination of proteinsextracted from erythrocyte ghosts. In contrast to proteinsfrom normal erythrocyte membranes, those extracted at lowionic strength from membranes of patients with spherocytosisresist aggregation when the ionic strength is increased (7).Others have shown that microfilaments with ultrastructuralcharacteristics similar to actin are generated from proteinspurified from erythrocyte membranes when ionic strength isincreased with divalent cations (8). Based on these observa-tions, we have suggested that defective formation of suchmicrofilaments might explain the increased rigidity and,thereby, the diminished survival of the hereditary spherocyte(7). Support for this supposition comes from accumulatedevidence that the shape and motility (or "contractility") ofcells derive from structural elements such as microtubules andmicrofilaments (9). The molecular characteristics of thesemorphologic elements allow a useful hypothesis: that is,structural proteins are a class of similar, but not identical,molecules (10). Furthermore, there are indications that themicrofilament proteins that may underlie cell shape share

471

important properties-for example, binding of myosin andprecipitation by alkaloids such as vinblastine, colchicine, andstrychnine-with molecules responsible for motility, such asactin filaments of muscle and the microfilaments involved incytoplasmic streaming (11, 12). Our recent observation (7)that protein extracted from hereditary spherocyte membranesprecipitates less readily than does normal membrane proteinwhen it is treated with vinblastine supports the possibilitythat defective membrane microfilaments might lead to theintrinsic rigidity and peculiar shape of the hereditary sphero-cyte. This paper provides further evidence to support thisconcept.We have used the rather specific capability of three diverse

compounds (vinblastine, colchicine, and strychnine) to pre-cipitate microfilament proteins to provide further proof thathereditary spherocytosis reflects an inborn defect in this classof proteins. All three compounds reversibly precipitate micro-filament proteins extracted from such disparate sources ascilia, mitotic spindles, erythrocyte membranes, and skeletalmuscle (actin) (13). In all situations, precipitation is reversedby removal of the drug by dialysis. Our studies indicate thatbrief exposure of normal erythrocytes to any of the threecompounds generates cells identical to the hereditary sphero-cyte.

MATERIAL AND METHODS

Heparinized blood was obtained from normal volunteers andthree unrelated spherocytic donors (two of whom weresplenectomized). Erythrocytes were washed three times inphosphate-buffered isotonic saline (pH 7.4; [P04] = 12 mM;[glucose] = 1i mM) before use. Vinblastine, colchicine, andstrychnine* were dissolved in isotonic saline and added at theappropriate concentration to a 30% suspension of erythro-cytes in phosphate-buffered saline, Unless otherwise stated,the final concentration of vinblastine was 0.2 mM; com-pletely analogous results were obtained with roughly 10-times higher concentrations of colchicine and strychnine.The loss of erythrocyte membrane through "budding" wasquantitated by analyses of total membrane lipid (mainlyphospholipid and cholesterol) during incubation (3, 4).Erythrocyte rigidity was estimated by the resistance of thecells to filtration by a modification of the paper-filtration

* Vinblastine sulfate was obtained from Eli Lilly Co., Indianap-olis, Ind.; colchicine and strychnine were obtained from SigmaChemical Co., St. Louis, Mo.

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472 Medical Sciences: Jacob et al.

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FIG. 1. Production of microspherocytes by vinblastine. Normal washed erythrocytes (left) become dense microspherocytes after 1hr of incubation with 0.2 mM vinblastine (right). No volume change accompanies the shape alteration.

technique described by Teitel (14). Briefly, a 50% suspensionof washed erythrocytes was filtered through a previously-moistened filter paper (Schleicher and Schuell, 7-cm diameter,white ribbon no. 589). The filter paper was supported in a

glass funnel, which was used throughout the studies. The rateof filtration was measured by continuously collecting thefiltrate in a tared vessel placed on a Mettler balance pan.

Weights of filtrate at 30-sec intervals were plotted on semi-logarithmic paper to give a straight line. The rate of filtrationis directly proportional to erythrocyte plasticity (i.e., in-versely proportional to erythrocyte rigidity). Erythrocytesurvival and organ sequestration studies of cells exposed todrugs were performed with Na25' CrO4-labeled erythrocytes as

described (15). Unreacted vinblastine, colchicine, or strych-nine was removed by a rapid wash in a 100-fold excess ofsaline just before injection of the cells.The ultrastructure of drug-treated erythrocytes was ex-

amined by scanning electron microscopy with a Cambridgestereoscan electron microscope, after preparation of the cellsin 1% glutaraldehyde. Transmission electron micrographswere prepared (16); thorium dioxide was used as an electron-dense -surface marker.

RESULTSBrief exposure to vinblastine, colchicine, or strychnine re-

produces in normal erythrocytes all the known in vitro and in

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FIG. 2. Increased osmotic fragility and loss of membranefragments (lipid) of erythrocytes incubated with vinblastine.After 1 hr of incubation with two different doses of vinblastine(open symbols), osmotic fragility-of normal erythrocytes increasessignificantly (left); no lipid (membrane) loss is detected. Afterprolonged incubation (right), membrane fragment loss (withouthemolysis) is shown by lipid depletion of erythrocytes. (left):* -4*, no vinblastine; O-O, 0.6 mM; A A 1.2 mM.

vivo characteristics of hereditary spherocytes. Results with allthree drugs were identical, except that 10-times more col-chicine and strychnine were required than vinblastine. Toconserve space, we present only the results with vinblastinehere.Normal erythrocytes exposed to vinblastine for 1 hr be-

come morphologically identical to hereditary spherocytes(right, Fig. 1). Microspherocytosis occurs without volumechange (as measured by hematocrit) or external membraneloss (as measured by lipid content). These cells are of increasedosmotic fragility (left, Fig. 2.); the fragility curves are char-acteristic of those observed in the genetic disease. Althoughthe effect is not detected in short-term experiments, afterovernight incubation as much as 20% of the membrane lipidis lost from treated erythrocytes into the medium (right, Fig.2). As in spherocytosis, the lipid moieties (phosphatides andcholesterol) are lost in exact proportion to their content inthe fresh cell. Thus, "buds" of membrane are lost duringprolonged incubation of treated cells exactly as observed inhereditary spherocytosis (3, 4).Another of the characteristics of hereditary spherocytes is

their increased permeability to sodium. A concomitant in-crease in active pumping of the cation, with an appropri-ately increased glycolytic rate, follows (1). These abnor-malities are reproduced in normal erythrocytes that are brieflyexposed to vinblastine, colchicine, or strychnine. Influx andefflux rates (measured with 24Na) are roughly doubled in

TABLE 1. Reversible induction of rigidity invinbiastine-treated erythrocytes*

RigidityFiltration (times

Erythrocytes half-time normal)

(min)Normal range (10) 1.2-2.0 1.0Hereditary spherocytes (3) 4.0-7.8 2.5-4.9Vinblastine-treated (3) 3.2-4.0 2.0-2.5

after dialysis (3) 1.0-2.0 0.6-1.3

* The rates of filtration through moistened filter papers ofwashed erythrocyte suspensions (50%) from normal volunteersand three patients are compared. Erythrocytes treated for 1 hrat 370C with 0.2 mM vinblastine were similarly filtered in threeexperiments before and after 4 hr of drug-removal by dialysis."Rigidity" is estimated by comparison of filtration half-timeswith those of normal erythrocytes.

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Erythrocyte Membranes and Spherocytosis 473

treated erythrocytes; concomitantly, glycolytic rates in-crease 20%. This increase is prevented if active pumping ofsodium is inhibited by ouabain.The rigidity of hereditary spherocytes is increased; the

half-time of their filtration through paper filters is roughly 3to 5 times that of normal erythrocytes (Table 1). Exposure ofnormal erythrocytes to vinblastine (or colchicine or strych-nine) for 30 min produces increases in erythrocyte rigidity(decreases in filtration rate) comparable to those observed withhereditary spherocytes. After removal of the drug by dialysisfor 3 hr, the erythrocytes regain normal plasticity, as shownby normalization of their filtration rates (Table 1).The most unique characteristic of hereditary spherocytes in

vivo is their specific entrapment and destruction in the spleen.When injected into normal subjects, vinblastine-treatederythrocytes labeled with 5"Cr behave identically (Fig. 3).Rapid removal of labeled erythrocytes (upper left, Fig. 3) isaccompanied by specific uptake of the label by the spleen(lower left, Fig. 3). However, as with hereditary spherocytes,these cells survive normally when injected into a splenecto-mized patient, and no evidence of hepatic sequestration isfound (right, Fig. 3).The fact that the rapid change in shape, and concomitant

increase in osmotic fragility, of vinblastine-treated erythro-cytes (Figs. 1 and 2) occur without volume change suggeststhat these cells lose membrane. However, at times whenspherocytosis is well-marked, no external loss is demonstrableby assay of lipid content of the treated cells. This seemingparadox is solved by ultrastructure studies. Scanning electronmicroscopy of vinblastine-treated erythrocytes demonstratesthe emergence of numerous cup- and bowl-shaped cells afterabout 30 min of exposure (left, Fig. 4). These shapes suggestthat invagination of membrane is occurring by a "purse-string" type of contractile process. When opposing edges ofuninvaginated membrane are resealed, more perfect micro-spherocytes are fashioned, some still containing dimples whereinvaginations originally occurred (left, Fig. 4). Sections oftreated erythrocytes viewed by transmission electron micros-

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FIG. 3. Specific splenic destruction of vinblastine-treatederythrocytes. Human erythrocytes treated for 1 hr with 0.2 mMvinblastine and labeled with 5"Cr disappear rapidly wheninjected into the normal circulation (left upper) and specificallyaccumulate in the spleen (left lower). In a splenectomized sub-ject (right), these same cells survive normally and without hepaticsequestration (lower) * , spleen; 0 O. liver.

copy, with thorium dioxide as an electron-opaque marker ofthe cell surface, makes the invagination process more obvious(right, Fig. 4); the endocytotic process is further demonstratedby the observation that intracellular vacuoles contain surfacematerial (thorium dioxide).

DISCUSSIONThese studies expand to human erythrocytes the conceptthat membrane microfilament proteins are crucial to normalcellular shape and plasticity. Furthermore, the observationthat chemical modification of this class of proteins reproducesall the known characteristics of hereditary spherocytes allowsthe reasonable conclusion that hereditary spherocytosis re-

FIG. 4. Scanning and transmission electron micrographs of vinblastine-treated erythrocytes. Left: Note bowl- and cup-shapedforms, especially along right border of this scanning electron micrograph; this suggests membrane invagination by a "purse-string"effect. More perfect spherocytes, some with dimples, are noted in the center of the micrograph. Right: The external surface of theerythrocyte is marked by electron-opaque thorium dioxide (small black dots). Note membrane invagination and endocytotic vacuolescontaining the opaque marker. The amorphous black clump on the left border of the invagination is an artifact.

Proc. Nat. Acad. Sci. USA 69 (1972)

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474 Medical Sciences: Jacob et al.

sults from mutations in microfilament proteins. The threealkaloids used in these studies, although significantly dif-ferent in molecular structure, share the ability to reversiblyprecipitate various microfilament proteins purified from di-verse types of cells (13). The effect of all three drugs is po-tentiated by calcium, protein precipitation being markedlyenhanced in the presence of this cation (13). It is of interest,therefore, that Ca++ has recently been demonstrated toproduce rigid spherocytes from normal erythrocytes (or theirghosts) when it accumulates intracellularly (17). As with thealkaloid drugs used in the present studies, this effect of Ca++is reversed by removal of the perturbing cation. We suggestthat calcium and the alkaloid drugs used herein act on iden-tical microfilament proteins in the erythrocyte membrane toreversibly alter their conformation in such a way as to resultin cell rigidity.We also note a further identity between the characteristic

properties of the alkaloids to precipitate purified microfila-ment proteins and their capacity to induce spherocytosis ofintact erythrocytes. That is, 10-times more colchicine andstrychnine are required than vinblastine both to precipitatepurified microfilmentous proteins (e.g., actin) (13) and togenerate spherocytes. Furthermore; the solubilization bydialysis of alkaloid-precipitated, purified protein finds an-alogy with the reversal of rigidity in vinblastine-treated eryth-rocytes after removal of the drug by dialysis (Table 1). Thereversibility of the rigidification process probably explainsour observations that only some vinblastine-treated erythro-cytes are rapidly removed from the circulation by the spleen;cells that survive for 8 hr or longer then survive perfectlynormally without evidence of further splenic entrapment. Wepresume that the drug effect has been "washed-away" duringcirculation for several hours in a drug-free environment.Whether a single molecular species of microfilament pro-

tein, or several such proteins, cause normal erythrocyte shapeand plasticity is not known. However, we do have preliminaryevidence (18, 19) that different mutations in membrane pro-tein are found in different families with the spherocytosis syn-drome. It is our view that any mutation that inhibits theaggregation into microfilarnents of protein subunits of thisclass might result in a rigid, and thereby, poorly viableerythrocyte. This view is in concert with the variable severityof the disease in different families.These results have provoked us to examine the effect of the

alkaloid drugs on other intact cell membranes. Vinblastine is

commonly used as a chemotherapeutic agent for variousmalignancies, especially the leukemias. The amount of thisdrug required to stiffen erythrocytes is more than 10-timeshigher than that probably present in the treated patient.Nonetheless, it will be of interest to assess the rigidity (andthus the motility) of cells such as granulocytes in vinblastine-treated patients. For example, it would seem especially dis-advantageous for patients with leukemia and, consequently,diminished numbers of normal granulocytes, to have themobility of these cells inhibited (e.g., into areas of bacterialgrowth).

Supported in part by research grants from the National In-stitutes of Health, USPHS (HE 12513) and from the Universityof Minnesota Graduate School. The capable laboratory assistanceof Miss Doris Kurth is gratefully acknowledged.1. Jacob, H. S. & Jandl, J. H. (1964) J. Clin. Invest. 43, 1704-

1720.2. Bertles, J. F. (1957) J. Clin. Invest. 36, 816-824.3. Weed, R. I. & Reed, C. F. (1966) Amer. J. Med. 41, 681-

698.4. Jacob, H. S. (1967) J. Clin. Invest. 46, 2083-2094.5. LaCelle, P. L. & Weed, R. I. (1969) Blood 34, 858.6. Barnhart, M. I., White, B. C., Singleton, E. M. & Lusher,

J. M. (1970) Proc. Int. Congr. Hematol., 13th 146.7. Jacob, H. S., Ruby, A., Overland, E. S. & Mazia, D. (1971)

J. Clin. Invest. 50, 1800-1805.8. Marchesi, V. T. & Steers, E., Jr. (1968) Science 159, 203-

204.9. Porter, K. R. (1966) in "Cytoplasmic Microtubules and

Their Functions," Ciba Foundation Symposium on Prin-ciples of Biomolecular Organization, eds. Wolstenholme, G.E. W. & O'Connor, M. (Little, Brown, and Co., Boston,Mass.), pp. 308-345.

10. Adelman, M. R., Borisy, G. G., Shelanski, M. L., Weisen-berg, R. C. & Taylor, E. W. (1968) Fed. Proc. 27, 1186-1193.

11. Hatano, S. & Tazawa, M. (1968) Biochim. Biophys. Acta.154, 507-519.

12. Puszkin, S. & Berl, S. (1970) Nature 225, 558-559.13. Wilson, L., Bryan, J., Ruby, A. & Mazia, D. (1970) Proc.

Nat. Acad. Sci. USA 66, 807-814.14. Teitel, P. (1967) Nouv. Rev. Fr. Hematol. 7, 195-214.15. Jacob, H. S. & Jandl, J. H. (1962) J. Clin. Invest. 41, 1514-

1523.16. White, J. G. & Davis, R. B. (1969) Amer. J. Pathol. 56, 519-

531.17. Weed, R. I., LaCelle, P. L. & Merrill, E. W. (1969) J. Clin.

Invest. 48, 795-809.18. Jacob, H. S., Overland, E., Ruby, A. & Mazia, D. (1970)

Blood 36, 841.19. Jacob, H. S., Overland, E., Ruby, A. & Mazia, D. (1970)

J. Lab. Clin. Med. 76, 885.

Proc. Nat. Acad. Sci. USA 69 (1972)

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