CHAOS CHAOS
J . M . M A R S H A L L
Department of Anatomy, School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania
I t is often assumed t h a t t h e t r a n s p o r t mechanisms which m a i n t a i n t h e internal environment of t h e cell and provide the m a t e r i a l s required
for growth and activity are localized directly in t h e cell m e m b r a n e . This concept is t a k e n for granted in most discussions of cell physiology and is in fact supported by a considerable body of indirect evidence.
Electron microscopists h a v e therefore looked for specialized s t r u c t u r a l features in the outer m e m b r a n e s of cells which might be e q u a t e d with the pores, p u m p s , and shuttling devices inferred from physiologi-
cal d a t a . T h e search h a s n o t been v e r y rewarding, however, and as a result there h a s been increased interest in more d y n a m i c concepts of m e m b r a n e structure and function. These are a t two different levels, and have in t h e p a s t occupied workers in r a t h e r different disciplines.
On the one hand, there has been discussion regarding molecular fluc- tuations, statistical pores, and specific carrier cycles—all representing d y n a m i c events a t or near the molecular level—which are usually conceived to occur within a larger static framework, " t h e cell m e m - b r a n e . " On t h e other h a n d are considerations of m e m b r a n e u p t a k e and renewal, of d y n a m i c exchanges occurring on a somewhat larger scale, as, for example, in pinocytosis and phagocytosis.
E v e n though we infer from electron microscopy t h a t m e m b r a n e movements and u p t a k e m u s t occur in a wide v a r i e t y of cells, t h e physiological implications of such activity r e m a i n uncertain because of t h e difficulty of obtaining q u a n t i t a t i v e information. F o r reasons which are chiefly technical, the free-living amoebae are t h e only cells for which we have estimates of the r a t e s of m e m b r a n e u p t a k e and of t h e a m o u n t s of w a t e r and solutes t r a n s p o r t e d under reasonably 1
Supported by Public Health Service Research Grant CA-01957 from the National Cancer Institute and by Research Career Development Award 5-K3-GM 477 from the National Institute of General Medical Sciences, of the United States Public Health Service.
33
34 J . M. MARSHALL
defined conditions, and some idea of the intracellular events which follow membrane ingestion.
In this chapter, we shall review the main features of membrane uptake and intracellular transformation in the giant amoeba Chaos chaos, and shall then describe what is known about the system as a whole in relation to ionic and osmotic regulation during normal growth. The work on which this account is based was done in collabo- ration with Dr. Carl Feldherr, Dr. Vivianne Nachmias, and D r . D a v i d Bruce, at the University of Pennsylvania. While the specific results m a y be relevant only to the free-living amoebae, certain concepts derived from the analysis of the system as a whole m a y provide a key to some of the broader problems of transport physiology.
T H E C Y C L E O F M E M B R A N E U P T A K E A N D R E N E W A L
I n Chaos ohaos, both pinocytosis and phagocytosis seem to depend upon the same fundamental mechanisms, and the vesicles formed in both processes undergo apparently identical transformations within the cytoplasm. The rate at which the cycle operates is not fixed, but varies greatly according to the physiological state of the cell.
In fasting cells under normal environmental conditions the turnover of surface membrane occurs only at a very low rate, as we shall see, even though the cells are actively moving and continually chang- ing shape. A stimulus to the surface coat of the membrane by a suitable food organism, or by the application of any of a variety of cationic-inducing substances (including small cations in appropriate concentrations, and positively charged proteins, dyes, or colloidal par- ticles) sets off the active process of membrane engulfment. Pinocytosis channels or food cups are formed, the continuity of the vesicle mem- brane with the surface membrane is broken, and continuity is reestab- lished in both by what seems to be an instantaneous reaction.
The surface membrane or plasmalemma of the amoeba is a com- posite structure, consisting in fixed specimens of an inner trilaminar or "unit" membrane about 100 Â thick, an intermediate amorphous coat about 300 Â thick, and an outer filamentous "fringe" which is about 1000 Â in thickness [11, 3, 2 ] . The coat material consists in the main of an acid mucopolysaccharide, containing about 5 % of sulfate, and the sugar is a polymannose. The slime coat has been shown to contain the binding sites for the cationic inducers of pinocy- tosis, and to play an analogous role in phagocytosis [ 6 ] . Its cation exchange capacity has been demonstrated [9]. Unpublished work sug-
gests t h a t it is t h e physical s t a t e of t h e slime coat which controls the osmotic permeability to w a t e r of t h e composite m e m b r a n e .
T h e r a t e of m e m b r a n e u p t a k e a n d t h e a m o u n t s of water, food, a n d various solutes t a k e n u p u n d e r defined physiological conditions have been estimated for C. chaos on t h e basis of m e a s u r e m e n t s on single cells a n d mass cultures [ 9 ] . T h e most useful condition to con- sider is t h e q u a s i - s t e a d y s t a t e of optimal growth. I n 24 hours, during which time t h e cell doubles in mass, t h e average amoeba consumes a b o u t 150 p a r a m e c i a . These are digested intracellularly in food vacuoles or vesicles, and t h e products of digestion are absorbed a n d utilized as t h e sole source of m a t e r i a l s for synthesis and energy m e t a b - olism. T a k i n g as a s t a n d a r d u n i t of growth t h e period required to double t h e cell mass, a n d considering t h e system to operate uniformly a n d continuously (an assumption which can be shown to be justified for t h e present purpose) we find t h a t 7 0 % of t h e cell surface m e m - b r a n e is t a k e n in per hour. T h e corresponding fluid u p t a k e is 4 0 % of average cell volume per hour. T h e n e t increase in cell volume is only 3 % per hour, a n d the bulk of t h e w a t e r t a k e n u p during feeding is excreted b y t h e contractile vacuole system, which keeps pace with n o r m a l v a r i a t i o n s in fluid u p t a k e to m a i n t a i n t h e w a t e r content of t h e ground cytoplasm a t a v e r y n e a r l y c o n s t a n t level.
T h e r a t e of a c t i v i t y of t h e system during r a p i d growth m u s t be compared with t h e two extremes, the r a t e of u p t a k e in t h e fasting or " b a s a l " s t a t e , a n d t h e r a t e during brief periods of m a x i m a l s t i m u l a - tion, to d e m o n s t r a t e t h e range of homeostatic regulation.
I n t h e absence of a n y specific stimulus to feeding or to pinocytosis, t h e cycle of m e m b r a n e t r a n s p o r t is s h u t down almost completely, even t h o u g h t h e cells are actively moving. T h e a m o u n t s of fluid a n d of m e m b r a n e t a k e n in are so low t h a t it is difficult to estimate t h e m , b u t we believe t h a t t h e y come to a b o u t 1% per hour of cell volume a n d surface area. Cells in t h e basal or fasting s t a t e lose weight by a b o u t 6% per d a y [ 1 2 ] , b u t can survive for 2 weeks or more.
T h e m a x i m u m r a t e of m e m b r a n e u p t a k e occurs under conditions which cannot be m a i n t a i n e d for more t h a n a few minutes, b u t properly prepared amoebae can feed very r a p i d l y for a b o u t 5 minutes, a n d in t h a t period will consume an area of m e m b r a n e equivalent to t h a t of their entire surface. ( T h e y m u s t p a y out new m e m b r a n e r a p i d l y in order to do this, a n d we shall r e t u r n to t h a t problem a little later.) D u r i n g t h e burst of activity, t h e r a t e of m e m b r a n e u p t a k e is a b o u t 17 times greater t h a n t h a t during optimal growth, or 1200 times t h e
36 J . M . MARSHALL
basal r a t e . After such a burst of activity, the cell requires a long period to restore t h e internal balance of the steady growth s t a t e . D u r i n g n o r m a l feeding a n d growth, t h e u p t a k e of surface m e m - b r a n e is closely m a t c h e d by t h e formation of new surface m e m b r a n e . T h i s process has been studied by labeling techniques a n d electron microscopy by D r . N a c h m i a s . I t appears t h a t m e m b r a n e expansion occurs by the interpolation of both lipid a n d mucopolysaccharide m a - terials directly from the ground cytoplasm, r a t h e r t h a n by t h e splicing in of preformed cytoplasmic m e m b r a n e s . T h i s is a key issue in t h e analysis of the system as a whole, because it follows t h a t the ingested m e m b r a n e , given a r a t e of u p t a k e of 7 0 % of the cell surface per hour, m u s t either a c c u m u l a t e progressively within t h e cell or be broken down into a dispersed, p r e s u m a b l y micellar, state and reutilized. Ac- cumulation of this order clearly does not occur, nor can t h e m e m b r a n e t a k e n up be accounted for by the very small fraction lost in defecation balls. F r o m such balance considerations it m u s t be concluded t h a t the greater p a r t of the m e m b r a n e ingested is broken down intracellu- larly. F r o m other studies which we shall not a t t e m p t to describe, it has been estimated t h a t the intracellular pool into which m e m b r a n e disappears and from which new m e m b r a n e forms comprises 9 to 1 0 times as much m a t e r i a l as is to be found in t h e surface m e m b r a n e itself a t a n y one time.
T h e pool from which m e m b r a n e forms a p p e a r s to be located in the ground cytoplasm, r a t h e r t h a n in a n y separate phase. W o r k i n g with p r e p a r a t i o n s of the ground cytoplasm isolated by direct centrifu- gation, we h a v e found t h a t m e m b r a n e s in the form of vesicles are readily formed de novo from optically clear p r e p a r a t i o n s . U n d e r slightly different conditions, similar p r e p a r a t i o n s of the ground c y t o - plasm will also form fibrils or microtubules in vitro, as shown by negative staining [ 9 ] . Although the vesicles, fibrils, and microtubules which a p p e a r in such p r e p a r a t i o n s are morphologically distinct, each m a y represent a slightly different s t a t e of t h e same lipoprotein m a t e r i a l .
T H E I N T R A C E L L U L A R T R A N S F O R M A T I O N S O F V E S I C L E S F O L L O W I N G U P T A K E
Before experimental studies h a d been done on t h e intracellular fate of pinocytic and phagocytic vesicles, one commonly heard two views expressed concerning t h e physiological significance of m e m b r a n e u p t a k e , both based largely upon a priori reasoning. According to t h e
first, vesicles passed through t h e cell, their m e m b r a n e s u l t i m a t e l y re-fusing with t h e surface m e m b r a n e . Their content was considered to be merely a p a r t of t h e external environment, t e m p o r a r i l y seques- tered, a n d their lining m e m b r a n e s not to be different in structure a n d function from the surface m e m b r a n e . Such a system, although it might be useful to transfer some m a t e r i a l s in bulk, seemed ill-suited to perform a n y of t h e highly selective functions which interest physiologists.
T h e a l t e r n a t i v e view was t h a t such vesicles represented a direct route of e n t r y into t h e cytoplasm. I t was postulated, for example, by B e n n e t t [ 1 ] , t h a t t h e vesicle m e m b r a n e s broke down and the con- t e n t of the vesicles was released into t h e cytoplasm. B y this means, according to its more enthusiastic p a r t i s a n s , pinocytosis provided t h e basic mechanism of active t r a n s p o r t .
Our experience with t h e amoeba has shown t h a t neither of these views is a d e q u a t e . T h e events which t a k e place within t h e cell follow- ing m e m b r a n e u p t a k e m u s t be described as a complex sequence of t r a n s f o r m a t i o n in both form a n d function. B y feeding into t h e system a v a r i e t y of different t r a c e r substances which can be detected by electron microscopy or by light microscopy, it has been possible to follow t h e m a r k e d vesicles for m a n y hours a n d to deduce something of t h e changes which occur within the cytoplasm.
Changes in Membrane Permeability C h a p m a n - A n d r e s e n and Holter showed t h a t
1 4
C - g l u c o s e is r a p i d l y metabolized once it enters the vesicles, even though it does not pass through the external cell m e m b r a n e [ 5 ] . I n parallel with this change in m e m b r a n e permeability, we have found t h a t there is a great i n - crease in t h e permeability of the m e m b r a n e to water. T o e s t i m a t e t h e m a g n i t u d e of t h e change, it was necessary first to determine t h e true p e r m e a b i l i t y to w a t e r of the external m e m b r a n e , i.e., t h e per- meability of t h e cell in t h e absence of active m o v e m e n t s of m e m b r a n e . T h i s is in fact a more difficult problem t h a n has been supposed, since most techniques do n o t distinguish between t h e e n t r y of w a t e r a t t r i b - u t a b l e to m e m b r a n e permeation and t h a t resulting from m e m b r a n e activity.
I n C. chaos, active m o v e m e n t s of m e m b r a n e are suppressed when the cell is cooled to 3 ° C . A t t h a t t e m p e r a t u r e t h e osmotic permeability coefficient is 10~
3
μ/αίτη/τηΊη, a value 10 times less t h a n t h a t obtained on active cells a t n o r m a l t e m p e r a t u r e ( L 0 v t r u p and Pigon, [ 8 ] ) . T h e
38 J . M . MARSHALL
difference is too great to be explained entirely by t h e effect of cooling on diffusion, a n d we therefore deduce t h a t t h e a p p a r e n t p e r m e a b i l i t y to w a t e r of t h e cell m e m b r a n e a t 2 5 ° C depends in p a r t on active m e m b r a n e movements, including t h e low basal level of pinocytosis a l r e a d y referred t o .
T h e t r u e p e r m e a b i l i t y to w a t e r of t h e surface m e m b r a n e in t h e amoeba is v e r y low, compared to t h a t of most cells; y e t t h e food vacuoles or vesicles undergo a r a p i d reduction in volume which results in a concentration of t h e vesicle content by more t h a n 10-fold. F r o m m e a s u r e m e n t s of m e m b r a n e area a n d of t h e volume changes in living cells, supplemented by t h e results of electron microscopy, we calculate t h a t t h e r a t e of shrinkage during t h e first 10 minutes implies a 100- fold increase either in p e r m e a b i l i t y or in pressure. T a k i n g into account t h e large initial size of t h e vesicles (up to 180 μ in diameter) and their irregular shape during the shrinkage process, there seems to be no mechanism capable of producing an increase in pressure of such m a g n i t u d e . W e therefore h a v e concluded t h a t t h e m e m b r a n e becomes v e r y much more permeable once it is t a k e n into the cytoplasm.
D u r i n g t h e period of rapid permeability change, t h e structure of the composite m e m b r a n e , as seen by electron microscopy, is modified.
T h e mucopolysaccharide coat, which initially lined the inner surface of t h e vesicle, breaks u p or is digested, b u t t h e u n i t m e m b r a n e p a r t of t h e composite structure appears unaltered.
The Changes Associated with Digestion
Over a period of hours, digestion t a k e s place within t h e food v a c - uoles. Changes in t h e structure of t h e individual particles of ferritin, when these are t a k e n u p in pinocytic vesicles, suggest t h a t digestion occurs within these vesicles as well. T h e r e is no direct evidence to indicate by w h a t route digestive enzymes enter t h e vesicular phase, but gold particles a n d ferritin, when injected directly into t h e ground cytoplasm, are subsequently found within digestive vesicles, even though t h e reverse m o v e m e n t does not occur. D u r i n g t h e digestive period, m a n y of t h e large vesicles divide, a n d there are m a n y fusions between vesicles as well. These can be d e m o n s t r a t e d by labeling vesi- cles with gold particles or ferritin. F r o m t h e morphological evidence, it appears t h a t additions to t h e p r i m a r y vesicles occur, n o t only by the fusion of the separate m e m b r a n e s of different vesicles, b u t also by t h e engulfment or enfolding of smaller vesicles within t h e p r i m a r y
vesicles. This process leads to the formation of complex polyvesicular bodies, but there is no difficulty in recognizing the relationships when labeling techniques are used.
The Fates of the Vesicular Membrane and of the Solutes Contained T h e membrane of the primary vesicle does not break down or disappear even though its permeability to water and to some solutes is increased, and even though a large p a r t of the membrane originally present is removed by a process we shall consider a bit later. When we trace the fate of vesicles containing ferritin or variously coated gold particles, it is invariably found t h a t particles of macromolecular size (about 100 Â) do not escape into the ground cytoplasm, even over periods as long as 96 hours.
During t h a t time the total surface area of the primary vesicle is greatly reduced. Reduction occurs by a remarkable process of "bud- ding" in which small secondary vesicles form singly or in short chains at the cytoplasmic surface of the primary vesicle. Whereas the pri- m a r y vesicles v a r y in diameter from 10 to 180 μ, approximately, the secondary vesicles are a b o u t 40 to 100 πΐμ in size. T h e process of budding a p p e a r s to be quite different from t h a t by which t h e p r i - m a r y vesicles are subdivided and fused on t h e larger scale. T h e secon- d a r y vesicles are n o t only m u c h smaller, b u t t h e y never contain t h e dense residual masses which are characteristic of the primaries, how- ever much t h e l a t t e r h a v e been subdivided or fused.
A p a r t from t h e differences in size a n d a p p e a r a n c e between the p r i m a r y and secondary vesicles, there are compelling reasons for con- sidering t h e two as different populations with different fates. T h e p r i m a r y vesicles evolve into dense bodies which are u l t i m a t e l y defe- cated, whereas t h e secondary vesicles m u s t u l t i m a t e l y disappear. T h e fraction of the m e m b r a n e originally ingested which remains with t h e p r i m a r y vesicle is n o t returned to t h e pool, b u t t h a t which is budded off to form secondary vesicles (some t h o u s a n d s of which are formed from each p r i m a r y vesicle) does r e t u r n to t h e cytoplasmic pool.
Solutes t a k e n u p in t h e original pinocytic or phagocytic vesicles are likewise p a r t i t i o n e d between t h e two p h a s e s — t h e p r i m a r y and secondary. M o s t t r a c e r substances fed into t h e system, in t h e experi- m e n t s we h a v e done, were retained in t h e p r i m a r y vesicles, a n d finally defecated. T h i s included ferritin, m e t h y l a t e d ferritin [10] a n d several t y p e s of gold sols t h e particles of which were coated or stabilized with different polyelectrolytes [ 7 ] . Such macromolecular particles did
40 J . M . MARSHALL
not a p p e a r a t all in t h e secondary vesicles even though t h e y were r a n d o m l y distributed when t h e p r i m a r y vesicles u n d e r w e n t t h e coarser t y p e of fusion a n d subdivision already described. T h e r e was, however, one interesting exception. Gold sol particles coated with p o l y a s p a r t i c acid behaved differently, in t h a t t h e y passed readily from t h e p r i m a r y into t h e secondary vesicles. W e h a v e no evidence to indicate w h y particles coated with polyaspartic acid a r e handled differently from particles of similar size b u t different surface chemistry, b u t t h e result suggests t h a t t h e content of t h e small secondary vesicles is determined by surface chemical forces a t work during t h e budding process. If this is so, t h e system as a whole should be thought of as one which is capable of separating specifically t h e solutes contained in t h e origi- nal vesicle, of determining which shall enter t h e secondary vesicles and u l t i m a t e l y t h e cytoplasm, a n d which shall be excluded from entry, and u l t i m a t e l y defecated.
I O N I C A N D O S M O T I C R E G U L A T I O N I N CHAOS CHAOS
F r o m t h e description a l r e a d y given, it should be evident t h a t a n y analysis of w a t e r a n d ion movements must t a k e into account n o t only t h e properties of t h e surface m e m b r a n e b u t also t h e functions of this d y n a m i c intracellular system.
D r . D a v i d Bruce a n d I have recently studied t h e relationships between t h e inside a n d outside levels of t h e principal ions of t h e cytoplasm, which in C. chaos are K, N a , a n d CI. D i r e c t analyses were done on samples of t h e ground cytoplasm, a n d t h e results were related to studies of electrical potential differences a n d of t h e resis- tance a n d rectifying properties of t h e surface m e m b r a n e under differ- ent conditions [ 4 ] . F o r t h e purpose of this volume, it seems a p p r o - priate t o consider only t h e general features of regulation suggested by t h e results.
I n t h e absence of active m e m b r a n e u p t a k e , t h e composite surface m e m b r a n e is essentially impermeable to anions b u t permeable t o both K
+
a n d N a +
, which exchange passively. I n t h e cold, when t h e con- tractile vacuole system as well as t h e system of m e m b r a n e u p t a k e is shut down, t h e cell does n o t discriminate between K
+
a n d N a +
. Under such conditions, it a p p e a r s t h a t t h e cytoplasmic cation level, the sum of [ K ]
in plus [ N a ]
i n, is determined b y a D o n n o n distribution, since t h e ground cytoplasm contains a n excess of anionic charges on the polyelectrolyte constituents, as well as Cl~ a n d some P 04" , a n d since t h e m e m b r a n e is essentially impermeable to all anions studied
so far. Because contractile vacuole a c t i v i t y is suppressed in t h e cold, the cell swells by a b o u t 1% of cell volume per hour, as w a t e r enters osmotically t h r o u g h the static surface m e m b r a n e .
W h e n t h e cell is r e t u r n e d to n o r m a l t e m p e r a t u r e s of 20° to 2 5 ° C , ionic a n d osmotic regulation are restored, as t h e two systems of m e m - brane u p t a k e and contractile vacuole o u t p u t become effective. I n t h e basal and active states described earlier, the cell m a i n t a i n s t h e compo- sition of t h e ground cytoplasm in respect to water, K
+
, a n d CI" by v a r y i n g t h e r a t e of contractile vacuole a c t i v i t y to m a t c h t h e v a r y i n g r a t e of u p t a k e . This is achieved b y excreting w a t e r with N a
+ and some anion not y e t determined (we suspect this m a y be P 04" , or some of the waste products of metabolism, or b o t h ) . As a result, t h e level of N a
+
in the c y t o p l a s m is n o r m a l l y very low (0.1 to 0.3 raM) compared to t h a t of K
+
(30 to 23 m M ) .
F r o m t h e electrochemical point of view, t h e complete system r e - quires a m i n i m u m of two active components of " p u m p s . " One is p r o - vided by t h e contractile vacuole subsystem, which m a i n t a i n s osmotic homeostasis a n d in doing so excretes N a
+
. T h e other is required to account for Cl~ accumulation against an a p p a r e n t electrochemical gradient.
W e know almost nothing a b o u t t h e molecular mechanisms which underlie t h e operation of t h e contractile vacuole system, b u t from morphological evidence t h e r e is little doubt t h a t the operation depends upon a series of intracellular t r a n s f o r m a t i o n s of vesicular elements, which a p p e a r de novo from the ground cytoplasm and fuse to form the contractile vacuole proper during each cycle of excretion. T h e over-all r a t e m u s t be a function of t h e r a t e a t which the smallest vesicles form from t h e ground cytoplasm, a n d this in t u r n m u s t be determined by t h e r a t e a t which w a t e r enters t h e cytoplasm, if we are to explain t h e range of homeostasis observed. I n this instance, the " p u m p " is clearly not a discrete mechanism localized in t h e surface m e m b r a n e , b u t a process based on t h e colloidal properties of t h e ground cytoplasm.
W i t h regard to t h e second p u m p i n g mechanism required by t h e electrochemical evidence, we h a v e come to a similar conclusion. T h e level of Cl~ in the cytoplasm (18 to 20 m M ) is m a i n t a i n e d even in the cold, and Cl~ is a c c u m u l a t e d during cell growth, y e t we find no evidence of a n y CI" p u m p in t h e surface m e m b r a n e . W e m u s t ask therefore whether t h e entire r e q u i r e m e n t for Cl ~ could be supplied by the route of pinocytic and phagocytic u p t a k e .
42 J . M . MARSHALL
Since we know the volume a n d ionic composition of t h e fluid t a k e n in during cell growth, t h e n u m b e r of food organisms consumed and the ion content of each, it is possible to d r a w u p a balance sheet.
T a b l e I gives t h e a m o u n t s of each of the three ions, N a +
, K +
, and Cl~, t a k e n in by an average-size amoeba (volume 33 X 10
6 /x
3
) during one growth cycle, during which the cell doubles in size. T h e total u p t a k e of each ion is compared with the requirement for each.
F r o m t h e table i t can be seen t h a t there is a reasonable correspond- ence between t h e t o t a l CI" u p t a k e and the a m o u n t required to double t h e cytoplasmic mass a n d volume. B o t h N a and Κ are also t a k e n up in a d e q u a t e a m o u n t s , b u t because these ions might also be supplied by passive exchange across t h e cell m e m b r a n e , we are less concerned
TABLE I. Ion Uptake Compared to Ion Requirements during the Growth of Chaos chaos, Feeding on Paramecium aurelia
a
Na Κ Cl
Ion content of fluid ingested 3 15 30--45
Ion content of paramecia ingested 14 95 14--18
Total u p t a k e
If
110 44--63Requirement for growth 1-2 90-100 60--67
α
Expressed in moles Χ 1 0
1 1
.
a b o u t their u p t a k e . W e conclude t h a t the amoeba is entirely dependent on m e m b r a n e u p t a k e to supply its requirement for chloride ion. T h e situation with regard to P 04
=
is p r o b a b l y similar, b u t this remains to be worked out.
T h e general concept suggested by the studies we h a v e described is t h a t all active t r a n s p o r t processes in the amoeba are intracellular and depend upon d y n a m i c transformations of m e m b r a n e into c y t o - plasm, and of cytoplasm into m e m b r a n e , r a t h e r t h a n upon the opera- tion of localized structures within an otherwise static "cell m e m b r a n e . "
T h e r e are several w a y s in which this concept can be further ex- plored in free-living cells, b u t it would be most interesting to deter- mine whether t h e t r a n s p o r t systems operating in higher organisms—
for example, in specialized epithelial tissues—are not similarly organized.
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