Organellar clusters formed by mitochondrial-rough endoplasmic reticulum associations: An ordered arrangement of mitochondria in hepatocytes

Organellar Clusters Formed by Mitochondrial-Rough Endoplasmic Reticulum Associations" An Ordered Arrangement of Mitochondria in Hepatocytes JOSEPH CASCARANO, PATRICIA A. CHAMBERS, EILEEN SCHWARTZ, PARVONEH POORKAJ, AND ROY E. GONDO

Our objective w a s to determine if mitochondrialr o u g h e n d o p l a s m i c reticulum (mt-RER) associations provide for an ordered a r r a n g e m e n t of m i t o c h o n d r i a in the cell. If such an ordered a r r a n g e m e n t exists, it might be manifested by g r o u p i n g of m i t o c h o n d r i a according to size and b i o c h e m i c a l properties. Liver h o m o g e n a t e was subjected to rate zonal centrifugation for fractionating mitochondrial clusters. These clusters w e r e t h e n exa m i n e d for morphological and biochemical characteristics. S c a n n i n g electron m i c r o s c o p y (SEM) s h o w e d that (1) m i t o c h o n d r i a w e r e held together in clusters by rough e n d o p l a s m i c reticulum, (2) clusters consisted of mitochondria of comparable size, and (3) a 45-fold difference in average mitochondrial v o l u m e existed b e t w e e n the organelles of the fastest and slowest s e d i m e n t i n g clusters. Transmission electron m i c r o s c o p y (TEM) affirmed that all of the organellar clusters e x a m i n e d w e r e mitochondria associated w i t h r o u g h e n d o p l a s m i c reticulum. C y t o c h r o m e oxidase and mitochondrial DNA w e r e found to be proportional to mitochondrial volume, indicating that these c o m p o n e n t s were s y n t h e s i z e d in proportion to increases in volume. Conversely, succinic dehydrogenase and ornithine c a r b a m o y l transferase were increased disproportionately (2.9-fold and six-fold, respectively) w i t h increase in mitochondrial volume. It is evident from this b i o c h e m i c a l h e t e r o g e n e i t y that clusters c o m p o s e d of larger m i t o c h o n d r i a differ functionally from clusters of smaller mitochondria. The size-ordered a r r a n g e m e n t suggests that this o r g a n i z a t i o n is in some w a y related to the biogenesis of h e p a t o c y t e mitochondria. It is also conjectured that the biochemical heterogeneity is a c o n s e q u e n c e of addition of selected proteins (e.g., succinic d e h y d r o g e n a s e and carbamoyl transferase) to m i t o c h o n d r i a in a d e v e l o p m e n t a l process as t h e y mature into larger organelles. (HEPATOLOGY 1995;22:837-846.)

Abbreviations: mt-RER, mitochondrial-rough endoplasmic reticulum; SEM, scanning electron microscopy; TEM, transmission electron microscopy; EDTA, ethylenediaminetetra-acetic acid; DAPI, 4'6'-diamidino-2-phenylindole. From the Department of Biology, University of California at Los Angeles, Los Angeles, CA. Received October 28, 1994; accepted March 30, 1995. Supported by University of California grant no. 2278. Address reprint requests to: Joseph Cascarano, PhD, Department of Biology, University of California at Los Angeles, Los Angeles, CA 90024-1606. Copyright © 1995 by the American Association for the Study of Liver Diseases. 0270-9139/95/2203-002253.00/0

Although mitochondria are generally thought of as being separate and distinct organelles, a number of studies have indicated that mitochondrial-rough endoplasmic reticulum (mt-RER) associations exist. In addition, evidence has been developed that suggests such associations involve all mitochondria in hepatocytes. At this juncture, it is not known if there is some intracellular order to these mt-RER associations. That is, (1) do these mt-RER associations form stable organellar clusters and (2) do such clusters differ from each other in terms of biochemical properties and size of component mitochondria? If these suppositions are correct, mt-RER clusters could be involved in carrying out different metabolic functions within the cell. Furthermore, the clustering of mitochondria of comparable size would imply that this organizational arrangement plays some role in the biogenesis of hepatocyte mitochondria. Fractionation of cellular homogenates by different centrifugation procedures has consistently resulted in co-sedimentation of some rough endoplasmic reticulum with mitochondria. TM In addition, mt-RER associations have been identified in a variety of cell types by electron microscopy. 5-9 It was subsequently shown in our laboratory that, in hepatocytes, mt-RER associations are extensive and in all probability involve the entire population of mitochondria. 1° Corroborating evidence for organellar associations was provided by scanning electron microscopy (SEM) of plasma membrane-denuded cells. ~'~2 Although the identity of some of the larger organelles visualized by SEM (nuclei and large mitochondria) was conjectured from transmission electron microscopy (TEM), many smaller organelles remained unidentified. ~2 Despite this evidence, the presence of mt-RER associations has been largely ignored. The existence of such associations is not of trivial consequence because they could provide order to the arrangement of mitochondria within the hepatocyte. No evidence has been developed that shows that an ordered mitochondrial arrangement exists. Morphometric analyses of TEM studies have been helpful in establishing numbers as well as the average volume of mitochondria in hepatocytes. 13'14 However, this approach has not provided any measure ofindividual mitochondrial volumes or of the actual range of mitochondrial volumes existing within the hepatocyte.

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If the networks of organelles visualized by SEM in plasma membrane-denuded cells are mitochondrial, their actual range of size is extensive indeed. Considering the possibility that substantive turnover of mitochondria occurs in hepatocytes, 1~ it should be anticipated that variability in organelle size would exist. In the event that mitochondria are clustered according to size, interesting implications are raised regarding their biogenesis in hepatocytes. Many studies have examined mitochondria for biochemical heterogeneity. These investigations were based on the fractionation of mitochondria by either density gradient or rate zonal centrifugation after a preparative differential centrifugation to remove "nuclei" and "post-mitochondrial particles. ''161s Although fractionation of mitochondria by rate zonal centrifugation is accomplished on the basis of differences in mitochondrial volume, no actual measurements of the size of these organelles were made. In addition, there was little or no awareness of the extensive association of mitochondria and RER. It is not clear if their homogenization procedures totally disrupted mitochondrial associations or if they were in fact fractionating mt-RER clusters. Nevertheless, most of the studies demonstrated mitochondrial biochemical heterogeneity. However, the physiological significance of these biochemical differences has remained obscure. Assuming that this biochemical heterogeneity exists, association of biochemically similar organelles into clusters would provide for the establishment of diverse metabolic domains within the hepatocyte. The objective of this investigation was to determine if mt-RER associations result in the formation of stable organellar clusters. If so, do such clusters have any special organization, i.e., are they composed of mitochondria of similar size? In addition, we wanted to determine the diversity of these organellar clusters in terms of size of component mitochondria, mt DNA content, and mt metabolic characteristics.

HEPATOLOGYSeptember 1995 zonal centrifugation 19 using a reorienting zonal rotor (SZ-14, Sorvall) and an RC2B centrifuge (Sorvall; DuPont Co., Wilmington, DE). The rotor was loaded at 3,000 r p m with 40 mL of sucrose cushion (60% wt/wt) and 1,250 mL of a linear sucrose gradient (14.5% to 39.5% wt/wt sucrose, 0.01 mol/L K-HEPES, pH 6.8) by means of a variable-speed Masterflex pump. The rotor was then accelerated to 10,000 rpm. Homogenate (25 mL) was loaded onto the linear sucrose gradient and centrifuged for 13 minutes. This was followed by a slow deceleration below 1,000 r p m (10 to 15 minutes) to allow for gradient reorientation. Thirty-two 40-mL fractions were collected and their turbidity measured at 700 n m in a Beckman DU spectrophotometer. Turbidity determinations were conducted to obtain a sedimentation profile of subcellular organel]es and to ascertain the distribution of mitochondria within the gradient (Fig. 1). Sucrose concentrations were determined using an Abbe refractometer (Fig. 1). This is a modification of a previously developed procedure. 2° To reduce the possibility of c o n t a m i n a t i o n by other organelles, fractions were pelleted a n d r e s u s p e n d e d as follows. E v e n - n u m b e r e d fractions from 10 to 30 were pelleted at 16,000 r p m for 15 minutes. These pellets were r e s u s p e n d e d in 8.5% wt/vol sucrose, 0.01 mol/L K - H E P E S p H 6.8 u s i n g a h a n d - d r i v e n 15-mL P o t t e r - E l v e h j e m glass-teflon homogenizer (10 strokes) a n d diluted to a final volume of 40 mL. These suspensions were t h e n centrifuged a t 11,000 r p m for 15 m i n u t e s a n d r e s u s p e n d e d in 13 m L of 8.5% wt/vol sucrose, 0.01 mol/L K - H E P E S as indicated. C e n t r i f u g a t i o n was rep e a t e d at 11,000 r p m for 15 minutes, a n d the final pellets were r e s u s p e n d e d in 4 m L of 8.5% wt/vol sucrose, 0.01 mo]/ L K - H E P E S pH 6.8 as indicated. These s a m p l e s were t h e n used for TEM a n d for enzyme assays. F o r DNA assays, pellets were r e s u s p e n d e d in 1.5 m L of 0.01 mol/L K - H E P E S , p H 6.8. Transmission Electron Microscopy. Selected fractions of

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MATERIALS A N D METHODS The following e x p e r i m e n t a l protocol was reviewed a n d approved by the I n s t i t u t i o n a l C o m m i t t e e on A n i m a l Welfare. Isolation o f Mitochondria. Male W i s t a r r a t s weighing app r o x i m a t e l y 300 g were a n e s t h e t i z e d w i t h an i n t r a p e r i t o n e a l injection of S u r i t a l (Parke-Davis, Morris Plains, NJ) (0.5 mg/ 100 g body weight) a n d decapitated. The livers were r a p i d l y removed, placed in ice-cold saline, weighed, sliced, a n d washed. Liver slices were finely minced by placing t h e m in a tissue p r e s s (1-mm d i a m e t e r holes) a n d slicing a w a y pieces 1 m m 3 or s m a l l e r as tissue was being extruded. To achieve r u p t u r e of cells w i t h m i n i m a l d i s r u p t i o n of t h e organelles, minced tissue was t h e n homogenized in 8.5% (wt/vol) sucrose, 0.01 mol/L K - H E P E S (pH 6.8) using a P o t t e r - E l v e h j e m glass homogenizer (Thomas Scientific, Swedesboro, NJ) a n d motordriven teflon pestle u s i n g only 2 strokes at 400 rpm. A p H of 6.8 was selected because studies on d e n u d e d cells demons t r a t e d a r e t e n t i o n of associations a t this pH. 12 The homogeh a t e (15% wt/vol) was t h e n filtered twice t h r o u g h two layers of nylon stocking m o u n t e d in a Swinnex filter (Millipore Corp., P h i l a d e l p h i a , PA). Fractionation of the homogenate was accomplished by rate

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FIG. 1. Sedimentation profile of liver homogenate subjected to rate-zonal centrifugation. Fractions of 40 mL were collected, assessed for % sucrose (wt/wt, A) and turbidity (0, absorbance at 700 nm). Fraction number is an inverse indicator of (1) distance from the top of the gradient and (2) the sedimentation velocity of the subcellular organelles in them. Fractions were pelleted, washed twice, resuspended in 4.0 mL sucrose (8.5% wt/vol) - K/HEPES (.01 mol/L, pH 6.8) and assessed for protein concentration (mg/mL, II). Washed mitochondria were used for all enzyme and DNA analyses. Each point is the mean of five separate experiments. The error bars indicate the standard errors of the mean when values exceeded the dimensions of the symbols.

HEPATOLOGYVo]. 22, No. 3, 1995 mitochondria, isolated as just described, were resuspended in 2% glutaraldehyde, 0.2 mol/L sodium phosphate, pH 6.8, 0.05 mol/L sucrose, and placed on ice for 1 hour. The mitochondria were then pelleted, the fixative decanted, and the mitochondria transferred to 1.5-mL polypropylene microcentrifuge tubes. Four washings by resuspension in 0.2 mol/L Sorenson's phosphate buffer, pH 7.421 and subsequent pelleting were followed by postfixation in 1% osmium tetroxide for 1 hour. The mitochondria were washed three times in 0.03 mol/L veronal acetate buffer, pH 5.922 and gently resuspended in several drops of ionagar (2% wt/vol in veronal acetate heated in a 70°C water bath). The mitochondria-ionagar suspension was then solidified by refrigeration and the microcentrifuge tubes removed with a razor blade. Mitochondrial blocks were trimmed into 1-mm cubes and stained en bloc in 1% wt/vol uranyl acetate (in veronal acetate) for 1 hour. After four washes in veronal acetate, the mitochondria were put through an ascending grade acetone dehydration (30% to 100%), infiltrated, and embedded in SPURR (Ted Pella Inc., Redding, CA). Polymerization was allowed to proceed for 24 hours at 60°C. Mitochondrial blocks were sectioned using glass knives and a Sorvall MT-2 ultramicrotome. Silver and gold sections were collected on 300-mesh copper grids and poststained in 26 mmol/L lead citrate 23 and 1% uranyl acetate. 24 Sections were then examined with a Zeiss EM 10 electron microscope. Scanning Electron Microscopy. To minimize disruption of the complexes, samples of mitochondria were taken directly from the fractions obtained after rate zonal centrifugation. Isolated mitochondrial complexes from different regions of the sucrose gradient were allowed to settle on millipore filters (Millipore Corp., 0.1 mm pore size) and fixed with 2% glutaraldehyde (Polysciences Inc.; Warrington, PA) that was dissolved in 0.1 moFL potassium phosphate buffer, pH 7.4. Filters with attached material were washed in double distilled water and transferred to 1% osmium tetroxide (Ted Pella Inc.) for 1 hour postfixation. After this, filters were washed several times in double distilled water and dehydrated through a graded series of ethanol (30% to 100%). Critical point drying with liquid CO2 was performed (Sandri pvt-3; Tousimis Research Corp., Rockville, MD) and the filters trimmed and secured to specimen stubs with double-stick tape. Mounted specimens were coated with gold-palladium in an argon filled vacuum sputter coater (Hummer I; Technics Inc., San Francisco, CA) and then examined with an Autoscan (ETEC Corp., Agawam, MA) electron microscope. 11 Random mitochondrial diameters were determined from random electron micrographs. Comparative mitochondrial volumes were estimated on the assumption that they were 1 spheres (V~phere = ~ ~dS). Enzyme Analyses. Succinate dehydrogenase activity was measured at room temperature by following the reduction of 2,6 dichlorophenolindophenol with a Cary 219 split-beam spectrophotometer at 600 nm. The reaction mixture contained potassium phosphate buffer, 50 mmol/L, pH 7.5; ethylenediaminetetra-acetic acid (EDTA), 1 mmoFL; 2,6 dichlorophenolindophenol, 1.66 mg/dL; potassium cyanide, i mmo]/ L; sodium succinate, 20 mmol/L; phenazine methosulfate, 0.3 mg/mL. An appropriate dilution of enzyme prepared in 0.01 mol/L potassium phosphate buffer was then added to the reaction mixture, giving a final volume of 420 #L. 2~ Cytochrome oxidase was measured spectrophotometrically at room temperature by following the rate of oxidation of reduced cytochrome c at 550 nm. 26 The reaction mixture contained 0.01 moFL potassium phosphate buffer, pH 7; 5 mg%

CASCARANO ET AL 839 (wt/vol) digitonin; and 50 #mol/L reduced cytochrome c. An appropriate dilution of enzyme prepared in 0.01 mol/L potasslum phosphate buffer was added to the reaction mixture to give a final volume of 420 #L. Glucose-6-phosphatase was measured by the appearance of liberated phosphate in the incubation medium. 27 The reaction mixture contained 25 mmol/L sodium cacodylate buffer, pH 6.5; 0.25 mmol/L EDTA; 25 mmol/L glucose-6-phosphate; and 50 ~L of mitochondrial sample, giving a final volume of 200 #L. The reaction was initiated with substrate addition and incubated at 37°C for 10 minutes. Addition of 4% (wt/ wt) ascorbic acid and 20% (wt/wt) trichloroacetic acid (5:2) stopped the reaction. The mixture was then placed on ice and subsequently centrifuged to remove the precipitate. Concentration of liberated phosphate was determined in 150 #L of supernatant with the addition of 75 ~L ammonium molybdate (1% wt/wt) and 150 #L of sodium arsenate-sodium citrate (2% wt/vol for each). Readings were taken 20 minutes later at 700 nm in a Beckman DU spectrophotometer. Ornithine carbamoyltransferase was measured by determining the appearance of citrulline in the incubation medium. The reaction mixture consisted of K-phosphate buffer, 67 mmol/L, pH 7.0; digitonin, 5 mg/dL; lithium carbamoyl phosphate, 12.5 mmoFL; L-ornithine, 12.5 mmol/L; and 50 #L of mitochondrial sample, to give a final volume of 200 #L. The reaction was initiated by the addition of substrate and was conducted at 37°C for 10 minutes. The reaction was stopped by addition of 250 ttL of HC104, 5%, and transferred to 0 to 4°C. The precipitate was removed by centrifugation, and citrulline was measured in the supernatants as described by Ceriotti. 2s DNAAnalysis. The DNA assay was a modification of a procedure described by Brunk et alY This assay uses DAPI (4',6'-diamidino-2-phenylindole), a dye that exhibits a 20-fold fluoresence enhancement on binding to AT-rich segments of DNA. Fluorescence was measured with a Farrand Spectrofluorimeter Mark I with corrected excitation module. The excitation and emission wavelengths were set at 340 nm and 448 nm, respectively, with slits at 5 nm on all ports without filters. Mitochondrial samples were subjected to three freeze-thaw cycles to insure accessibility of mitochondrial DNA to DAPI. Calf thymus DNA, 60 mg/mL (Sigma Chemical Company, St Louis, MO), was used as a standard. Aliquots of mt-RER suspension and of standard were incubated in the presence and absence of DNase I (Sigma Chemical Company). The DNase reaction mixture contained MgC12, 5 mmol/L; KHEPES, 8.33 mmol/L, pH 7.0, digitonin, 0.1%; DNase I, 100 U/mL; 245 #L of mt-RER suspension or of standard in a final volume of 300 #L. Incubation was for 1 hour at 37°C. These samples were then added to DAPI solution in the following way. Three mL of DAPI solution was added to a 1.0 cm × 1.0 cm × 4.5 cm quartz cuvette. The DAPI solution consisted of: DAPI, 100 ng/mL; NaC1, 100 mmol/L; EDTA, 10 mmol/L; K-HEPES, 2 mmol/L, pH 7.0. Three sequential 30#L aliquots of DNased or non-DNased mt-RER suspensions or standard were mixed into the DAPI solution. Fluorescence readings were taken before and after addition of each 30-#L aliquot to obtain the fluorescence enhancement of the added material. Fluorescence from sources other than DAPI-DNA binding were excluded by subtracting the fluorescence increases of the DNased sample from the fluorescence increases of the non-DNased sample. Protein. Protein was determined spectrophotometrically (700 nm) by a modified Lowry method at room temperature. Bovine serum albumin was used as the standard. 8° This pro-

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HEPATOLOGYSeptember 1995

We then wanted to determine if it was possible to retain clusters of mitochondria associated with RER when tissue was subjected to homogenization and fractionation by rate zonal centrifugation. To accomplish

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FIG. 2. Comparison of liver homogenates fractionated with two different gradients. Fractions of 40 mL were collected after centrifugation. Turbidity of each fraction was determined to assess the sedimentation profile of subcellular organelles. Turbidity is displayed as % of the maximum turbidity obtained in fraction 15. Gradient 1 (O): cushion, 150 mL 60% wt/wt sucrose, followed by 1,050 mL 45% (wt/ wt) to 14.5% (wt/wt) sucrose. Gradient 2 (i): cushion, 40 mL 60% (wt/wt) sucrose, followed by 1,250 mL 39.5% (wt/wt) to 14.5% (wt/ wt) sucrose. Gradient 2 allowed increased separation of organelles because of its more shallow density and increased volume. Gradient 2 is identical to conditions illustrated in Fig. 1.

cedure uses sodium dodecyl sulfate to enhance solubilization of membrane-bound proteins. RESULTS

Isolation and Fractionation of Mitochondrial-RER Clusters. For this investigation we modified the rate z o n a l g r a d i e n t p r e v i o u s l y u s e d . 4'2° T h e c u s h i o n w a s r e d u c e d f r o m 150 m L to 40 m L 6 0 % ( w t / w t ) s u c r o s e , 0.01 mol/L K-HEPES. The gradient was increased from 1,050 m L to 1,250 m L . I n a d d i t i o n to e x t e n d i n g t h e gradient, it was also made more shallow by changing s u c r o s e c o n c e n t r a t i o n s f r o m 14.5% w t / w t to 4 5 % w t / w t s u c r o s e to 14.5% w t / w t to 3 9 . 5 % w t / w t s u c r o s e . T h e effect of extending the gradient and making it more s h a l l o w p r o v i d e d a g r e a t e r a b i l i t y to i n c r e a s e t h e s p r e a d o f o r g a n e l l e s i n t h e g r a d i e n t . T h i s is e v i d e n t b y comparing the turbidity profiles of the collected fract i o n s f r o m b o t h t y p e s o f g r a d i e n t ( F i g . 2).

FIG. 3. SEM and TEM of an mt-RER cluster. After fractionation of liver homogenate, as indicated in Fig. 1, samples were recovered directly from fraction 15 and prepared for SEM and TEM. (A) Scanning electron micrograph of an mt-RER cluster. Mitochondria are clearly held together by membranous material. Ruptures in membranes holding mitochondria together are artifacts resulting from bombardment by the electron beam. (Original magnification ×26,600.) (B) Transmission electron micrographs prepared from mitochondrial pellets of fraction 15. Rough endoplasmic reticulum is interposed between mitochondria. (Original magnification ×56,000.)

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CASCARANO ET AL

this, homogenization was held to minimal disruption, as described under Materials and Methods, to reduce the shearing stress applied to mt-RER clusters. This homogenate was then subjected to rate-zonal centrifugation. Figure 3a is a SEM of a representative sample from the center of the gradient (fraction 15; see turbidity profile, Fig. 1). Clusters of organelles were seen held together by endoplasmic reticulum. These organelles were subsequently confirmed to be mitochondria and RER by TEM (Fig. 3b). Most of the mitochondria were seen associated with RER. With the retention of clusters through homogenization and centrifugation, we wanted to determine if the size characteristics of mitochondria in these clusters was ordered or haphazard. To determine if size characteristics of mitochondria in these clusters varied with sedimentation velocity, the experiment was repeated. Samples were taken from various regions of the gradient for examination by SEM (Table 1, Fig. 4). Clusters of organelles held together by endoplasmic reticulum were seen in all of the fractions examined. However, fraction 30 exhibited extensive contamination with membranous material. This fraction was pelleted and washed as described under Materials and Methods and reexamined by SEM. Clusters of organelles similar to those seen in other fractions were then visualized in fraction 30 (Fig. 4). Although individual mitochondria were seen occasionally by SEM, the vast majority of organelles (>90%) existed in clusters (Fig. 4). Even though mitochondrial diameters varied within a fraction, differences between fractions were far greater. A substantial difference in diameters was not evident between fractions 7 and 11, but significant diminutions occurred with decreasing sedimentation velocity of these mtRER clusters (Table 1). Although the organelles in fraction 15 (Fig. 3) were clearly recognized as mitochondria, we wanted to ascertain t h a t mitochondria were present in the other fractions studied. Mitochondrial pellets obtained from fractions 10 through 30 were prepared for TEM. There was insufficient material in fractions 7 through 9 for analysis by TEM. However, these TABLE 1. M i t o c h o n d r i a l S i z e R e l a t e d to Sedimentation Velocity Fraction

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NOTE. Diameters of mitochondria were obtained from scanning electron micrographs of mt-RER clusters obtained from different fractions of the gradient. Examples of micrographs are presented in Fig. 4. * Number of mitochondria measured. t Mean +_ SEM. $ V = 1/6~d 3.

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FIG. 4. Scanning electron micrographs of mt-RER clusters displaying different sedimentation velocities. Liver homogenate was fractionated as indicated in Fig. 1. The following samples were taken directly from the gradient after rate-zonal centrifugation: (A) fraction 7, (B) fraction 11, (C) fraction 15, (D) fraction 20, and (E) fraction 26. Sample (F) fraction 30 was taken from mitochondria that had been pelleted and washed as described in Fig. 1. Mitochondria in sample (A) were the fastest sedimenting organelles, whereas those in sample (F) were the slowest. (Original magnification ×8,000.)

studies clearly confirmed the presence of mitochondria in all of the other fractions examined (Fig. 5). Furthermore, average diameter of these sectioned profiles decreased with increasing fraction number, as was evident with SEM-examined organelles. Although some of the mitochondria were in the condensed state, others exhibited various stages of disruption of cristae. We believe this is the result of the shear stress in the double washing-resuspension procedure used. Remnants of endoplasmic reticulum (which are presumed to be RER) can be seen between some of the mitochondria. Biochemical Heterogeneity of Size-Fractionated mtRER Clusters. We then wanted to determine if a biochemical heterogeneity accompanied the mitochondrial size classes demonstrated in the previous section. Washed mitochondrial pellets were used for this aspect of the study. Examination of the fractions indicated in the sedimentation profile showed t h a t fractions 1 to 3

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contained red blood cells that had been trapped in the tissue vasculature (Fig. 1). Fractions 4 through 6 contained little detectable material. Although organelles were visualized by SEM in fraction 7 (Table 1, Fig. 4), the amount of material that could be recovered from fractions 7 through 9 was insufficient for meaningful enzymatic analyses. Figure 1 shows the protein recovery obtained in fractions 10 through 30 after the washing procedure. To assess for mitochondrial biochemical heterogeneity, two mitochondrial membrane enzymes, cytochrome

HEPATOLOGY September 1995

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Fraction Number FIG. 6. Cytochrome oxidase activity (#mole cytochrome with oxidized/mg protein × min). Analyses were conducted on mitochondria t h a t h a d been pelleted and washed after separation by rate-zonal centrifugation (see Fig. 1). Each point is the m e a n of five separate experiments. The error bars indicate the s t a n d a r d error of the means. The activities in the different fractions do not differ significantly, with the exception of 10, 16, and 30. This is a mitochondrial memb r a n e enzyme encoded by both mitochondrial and nuclear genomes.

FIG. 5. Transmission electron micrographs of mt-RER clusters displaying different sedimentation velocities. After liver homogenate was subjected to rate-zonal centrifugation, mitochondria were prepared for sectioning a n d electron microscopy following the pelleting and washing procedure indicated in Fig. 1. The fractions examined are represented as follows: (A) fraction 10, (B) fraction 14, (C) fraction 18, (D) fraction 26, a n d (E) fraction 30. Mitochondria are present in all fractions. Although some show the condensed configuration, others exhibit various stages of cristal disruption because of the shear stress in the double w a s h i n g - r e s u s p e n s i o n procedure. R e m n a n t s of endoplasmic reticulum can be seen interspersed between mitochondria. Mitochondria displayed in (A) were the fastest sedimenting organelles, whereas those in (E) were the slowest. (Original magnification ×30,000.)

oxidase and succinic dehydrogenase, and a matrix enzyme, ornithine carbamoyl transferase, were selected for study. To obtain a quantitative assessment of the presence of RER, analyses of glucose-6-phosphatase were conducted. Examination of the cytochrome oxidase-specific activities (Fig. 6) showed that fractions 10 and 30 were significantly lower than the activities in the other fractions. With the exception of 16, the remainder of the fractions did not differ significantly from each other. Glucose-6-phosphatase activity was fairly constant in fractions 10 through 22 and increased continuously thereafter, being more than 10 times greater in fraction 30 (Fig. 7). If we assume glucose-6-phosphatase is proportional to RER protein and that cytochrome oxidase is proportional to mitochondrial protein, the ratio of these two activities would provide a quantitative indication of the degree of association of mitochondria and RER. These considerations also point out that the RER presence contributed to error in determinations of cytochrome oxidase specific activities. The error introduced in fractions 10 through 24 would appear to be relatively minor b u t would increase progressively for fractions 26, 28, and 30. If we discount the lowered activity of fraction 30 on the basis of RER contamination, it appears that the proportion of cytochrome oxidase to total mitochondrial protein remains constant despite the large range in organelle size. The distribution of activities for succinic dehydrogenase differs from that of cytochrome oxidase. As mitochondria decrease in size, specific activity of succinic dehydogenase decreases (Fig. 8). This is not an illusory

CASCARANO ET AL

HEPATOLOGY Vol. 22, No. 3, 1995 1o0

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FIG. 7. Glucose-6-phosphatase activity (nm P~ releasecYmg protein × min). All conditions identical to Fig. 6. This enzyme was selected as a measure of the presence of rough endoplasmic reticulure. Activities increase significantly in fractions 26, 28, and 30. Each point is the m e a n of five separate experiments. The error bars indicate the s t a n d a r d errors of the mean. Inset: ratio of glucose-6-phosphatase activity to cytochrome oxidase activity (G6P/CO ratio). This serves to show t h a t smaller mitochondria have considerably greater association with rough endoplasmic reticulum. Consequently, specific activities of mitochendrial enzymes show increasing diminution as a result of increasing association with rough endoplasmic reticulure in fractions 26, 28, and 30.

20

0

I 24

I 26

I 28

J 30

Fraction N u m b e r

FIG. 8. Succinic dehydrogenase activity (nm 2,6 dichloro-phenolindophenol reduced/mg protein × min). All conditions are identical to Fig. 6. Activities increase significantly as mitochondria increase in size. Each point is the m e a n of five separate experiments. The error bars indicate the s t a n d a r d errors of the mean. Succinic dehydrogenase is a mitochondrial m e m b r a n e enzyme encoded by the nuclear genome. Inset: ratio of succinic dehydrogenase activity to cytochrome oxidase activity (SDH/CO Ratio). This ratio cancels any bias in the specific activities introduced by the large presence of rough endoplasmic reticulum protein present in fractions 26, 28, and 30. This shows t h a t succinic dehydrogenase is increased in greater proportion to cytochrome oxidase with increase in mitochondrial size despite the fact t h a t both are m e m b r a n e enzymes.

FIG. 9. Ornithine carbamoyl transferase activity (nm citrulline produced/mg protein × rain). All conditions are identical to Fig. 6. Activities increase significantly as mitochondria increase in size. Each point is the m e a n of five separate experiments. The error bars indicate the s t a n d a r d errors of the mean. Ornithine carbamoyl transferase is a matrix enzyme and one of the enzymes involved in the urea cycle. Inset: ratio of ornithine carbamoyl transferase activity to cytochrome oxidase activity (OCT/CO ratio). Ornithine carbamoyl transferase is increased in greater proportion to cytochrome oxidase with increase in mitochondrial size.

effect caused by increases in RER protein in fractions 26, 28, and 30. If we obtain ratios of specific activity for succinic dehydrogenase:cytochrome oxidase, the contribution of RER protein is cancelled, and the ratios show a continual decline from fraction 10 to fraction 30. Although these are both membrane enzymes, their relative proportion is altered 2.9-fold with mitochondrial size. In a similar vein, ratios of specific activity for ornithine carbamoyl transferase:cytochrome oxidase show a 6.1-fold decline from fraction 10 to fraction 30 (Fig. 9). It is evident that larger mitochondria are not simply proportional enlargements of smaller organelles. Certain components are increased disproportionately with size, indicating that larger mitochondria (>0.5 mm diameter) have metabolic functions that differ from those of smaller organelles. Last of all, we examined each of the fractions for DNA content (Fig. 10). The DNA/protein ratios for fractions 10 and 12 were higher than those in any of the other fractions. More importantly, all of the other fractions, 14 through 30, do not differ significantly from each other. In fact, fraction 12 differs only from three fractions; 20, 28, and 30. Two of these, fractions 28 and 30, have substantial RER protein present. The DNA/ protein ratios were divided by the specific activities of cytochrome oxidase to correct for the presence of RER protein. This ratio was 12.1 for fraction 12 and ranged from 9.3 for fraction 14 to 7.5 for fraction 30. The mean for fractions 12 through 30 was 8.1. It is evident that DNA is proportional to cytochrome oxidase (and therefore to mitochondrial protein) in fractions 12 through

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CASCARANO ET AL

HEPATOLOGYSeptember 1995

~6 a~ ~5

~4 Z ~3

10

I

I

I

12

14

16

I

I

I

18 20 22 Fraction N u m b e r

I

I

I

24

26

28

30

FIG. 10. Mitochondrial DNA content (#g DNA/mg protein). All conditions are identical to Fig. 6. Despite a 40-fold range in mitochondrial volume, the ratio of DNA to protein appears relatively constant, 1.84 (fraction 30) to 3.43 (fraction 14), with the exception of higher values in fractions 10 (6.37) and 12 (4.35). Inset: to eliminate the bias introduced by rough endoplasmic reticulum protein, the DNA/ protein ratio was divided by cytochrome oxidase activity (DNA/CO ratio). Fraction 10 is still substantially higher than all other fractions, perhaps as a result of contamination from nuclear fragmentation. The DNA/CO ratio is clearly a constant, ranging from 7.5 (fraction 30) to 9.3 (fraction 14), strongly suggesting that both of these components increase proportionately with mitochondrial size.

30. The only fraction not conforming to this relationship is fraction 10. It is possible that this is caused by contamination from fragmented nuclei. Considering that there is approximately a 40-fold range of mitochondrial volumes between fractions 12 and 30, it is evident that DNA content is not a constant but is proportional to organelle size. DISCUSSION

Before this study, the extent of diversity in hepatocyte mitochondrial size was not known. Except for the fact that mitochondria were found to be associated with rough endoplasmic reticulum, 1° there was no knowledge of any other aspect of mitochondrial organization in the cell. In this investigation we have discovered that (1) the range of mitochondrial volumes are extensive (at least 45-fold) and (2) mitochondria are not haphazardly arranged but are held together according to size by rough endoplasmic reticulum. This organization of mitochondria has made it possible for us to separate clusters according to the size of the component organelles by rate zonal centrifugation. Cytochrome oxidasespecific activities and the ratios of mitochondrial DNA:mitochondrial protein were found to be proportional to mitochondrial volume over essentially the entire size range of mitochondria. However, succinic dehydrogenase and ornithine carbamoyl transferase were increased disproportionately in clusters consisting of larger mitochondria. The latter clearly indicates that

some major biochemical differences exist between fractionated mt-RER clusters. The organellar membranous clusters visualized with SEM (Figs. 3 and 4) were deemed to be mitochondria and endoplasmic reticulum on the basis of the following criteria: Examination of fraction 15 (Fig. 3b) with TEM showed mitochondria associated with RER. Other studies have demonstrated essentially similar relationships between RER and mitochondria in TEM of isolated organelles. 4'1°'31 Furthermore, associations between mitochondria and RER are quite stable because isolated organelles subjected to EDTA or hypotonic-hypertonic t r e a t m e n t retain substantial associations) 1 Mitochondria with remnants of endoplasmic reticulum were present in all fractions (Fig. 5). Analyses conducted for mitochondrial and RER marker enzymes (cytochrome oxidase and glucose-6-phosphatase) provided affirmation for the presence of these organelles in all fractions examined. Glucose-6-phosphatase is present in both smooth and rough endoplasmic reticulum of liver) ~ With homogenization, smooth ER and RER (not associated with mitochondria) form vesicles (rough and smooth microsomes). These vesicles have sedimentation characteristics that result in their separation from mitochondria during centrifugation) ~ On this basis, we assumed that the glucose-6-phosphatase activity present in the mitochondrial pellets represented the degree to which mitochondrially associated RER was present. In a previous investigation, 1° we examined serial sections of liver to study the association of mitochondria and RER in situ. It was discovered that a saccule of RER remained associated with a particular mitochondrion through a series of sections. This indicated that the association existed over a broad area of the mitochondrial surface. Furthermore, RER saccules exhibited such communication with several mitochondria. From this, we developed a schematic model that approximates the organellar membranous clusters visualized with SEM in the current study. It should be noted that in situ associations have also been described by other investigators, s'~ Because cells other than hepatocytes (endothelial, Kupffer, fat-storing cells) are present in liver, consideration should be given to the contribution of their organelles to the results obtained. Although hepatocytes constitute approximately 70% of the liver's cellular population, their size is such that they constitute 93% of the liver's cytoplasmic volume. Morphometric analysis also projects that 98.8% of the liver's mitochondrial volume and 93% of its RER surface area exists in hepatocytes. 14Although mitochondria come almost completely from hepatocytes, it might be argued that the smaller organe]les are from the nonparenchymal cells (endothelium, etc.). However, examination of TEM studies of nonparenchymal cells show only occasional mitochondria scattered throughout the cytoplasmJ 4 No dense populations are evident as would be suggested by the types of clusters visualized in Fig. 2 (fraction 30). Furthermore, glucose-6-phosphatase is not detectable in nonparenchymal cells. 32 The smallest organelles that we isolated (fractions 26 through 30) dem-

HEPATOLOGY Vo]. 22, No. 3, 1995

onstrated increasing association with RER and glucose6-phosphatase. Therefore, we believe that the entire size range of mitochondria that we have visualized exists within hepatocytes. An important factor for consideration is that hepatocytes display a heterogeneous distribution in the liver lobule. For example, histochemical studies have shown that succinic dehydrogenase activities are considerably greater in periportal (zone 1) cells as compared with perivenous (zone 3) cells. Cytochrome oxidase and ornithine carbamoyl transferase have essentially the same distributions, exhibiting slightly greater activities in periportal cells. 33 Morphometric analyses show that total mitochondrial volume for periportal cells is essentially the same as that for pericentral cells in livers of rats 6 months of age or younger. 34 Because there are a greater number of mitochondria in perivenous cells, 14 the average organelle has a smaller volume than the average organelle in periportal cells. This should not be construed to mean that there is no large range of mitochondrial sizes and enzyme activity within these hepatocytes and that such ranges do not overlap for periportal and perivenous cells. The mitochondria we have isolated come from all hepatocytes in the liver lobule. If all cells have a variety of size ranges and enzyme activities, mitochondria will become interspersed on homogenization, and it would not be possible to designate which mitochondria originated in periportal cells and which originated in pericentral cells. This is supported by the disparate distributions of cytochrome oxidase and ornithine carbamoyl transferase activities in our isolated mitochondria (Fig. 9). Such a result would not be anticipated if all mitochondria within cells of each region were of uniform size and identical enzyme activity. These mt-RER associations led to the supposition that this subcellular organization might be involved in the synthesis of cytochrome P450. Studies with subcellular fractions 3~ and in situ labeling 36 have shown that although some minor synthesis of cytochrome P450 appears to occur in mitochondrially associated RER, the nonassociated RER is the major contributor to the synthesis of this protein. The functional significance of these mt-RER associations remains obscure. The clustering of mitochondria according to size is an important discovery. We are hypothesizing that the clusters of increasing sedimentation velocity (Table 1) result from a progression of mitochondrial growth. This raises the possibility that the RER is contributing to mitochondrial growth in some manner. Evidence indicates that mitochondrial proteins are synthesized on free polysomes and are imported posttranslationally from the cytosolF However, phospholipids are synthesized by the RER. 3s It has been suggested that soluble phospholipid exchange proteins in the cytosol shuttle phospholipids from RER to mitochondria. 3s Given that mitochondria and RER exist in clusters, the transfer of phospholipids to organelles would appear to occur directly. Considering that liver mitochondria are projected to have a half-life of 9.4 days 15 and that a prodigious number of organelles are present

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in hepatocytes, the mitochondrial-RER clusters provide an assembly-line mechanism for synthesis of these organelles. Increase of mitochondrial DNA in proportion to organelle size is another interesting discovery. No previous determinations of DNA content related to mitochondrial size exist. However, previous studies do indicate that considerable variability exists in the DNA content of m i t o c h o n d r i a 9 Examination of mitochondria for DNA-containing regions indicated that there were anywhere from two to six nucleoid regions in these organelles. In addition, more than one DNA molecule could be associated with one nucleoid. 39 Our study is not inconsistent with these findings. It is not unreasonable to assume that the variability in mitochondrial nucleoid regions and in numbers of DNA molecules associated with them is not an accidental phenomenon but is in all likelihood related to increases in mitochondrial volume (or growth). Biochemical heterogeneity of mitochondria has been demonstrated a number of times previously. However, the functional significance of this finding has remained obscure. From this study, some understanding of this phenomenon emerges. Of the three enzymes examined, only cytochrome oxidase has the same ratio of activity to volume in small (less mature) organelles as in large (or more mature) organelles. This suggests that synthesis of cytochrome oxidase occurs in proportion to increase in mitochondrial volume. Succinic dehydrogenase and ornithine carbamoyl transferase appear to be added disproportionately as organelles mature. Under such circumstances, heterogeneity would be related to the growth or maturation of the mitochondria. The end effect is that larger mitochondria are poised to conduct metabolic processes substantially different than those occurring in smaller, less mature organelles. For example, the distribution of ornithine carbamoyl transferase suggests that urea cycle involvement is relegated almost exclusively to larger organelles. This "developmental heterogeneity" may contribute importantly to the flexibility of hepatocyte metabolism in adjusting to major physiologic stresses such as variation in diet, starvation, etc. The clustering of organelles by RER also raises the possibility that this phenomenon creates microenvironments in the cell, facilitating transfer of metabolites between mitochondria and cytosol, as occurs in the urea cycle. REFERENCES 1. B r u n n e r G, Bygrave FL. Microsomal m a r k e r enzymes and their limitations in distinguishing the outer m e m b r a n e of r a t liver mitochondria from the microsomes. E u r J Biochem 1969;8:530534. 2. F r a n k e WW, Kartenbeck J. Outer mitochondrial m e m b r a n e continuous with the endoplasmic reticulum. Protoplasma 1971; 73:35-41. 3. Shore GC, Tata JR. Two fractions of rough endoplasmic reticulure from r a t liver. I. Recovery of rapidly sedimenting endoplasmic reticulum in association with mitochondria. J Cell Biol 1977;72:714-725. 4. Pickett CB, Montisano D, Eisner D, Cascarano J. The physical association between r a t liver mitochondria and rough endoplas-

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