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The membrane lattice: A novel organelle of the trypanosomatid flagellate Leptomonas collosoma
JOURNAL OF ULTRASTRUCTURE RESEARCH
72, 2 0 0 - 2 0 5
(1980)
The Membrane Lattice: A Novel Organelle of the Trypanosomatid Flagellate Leptomonas collosoma JEAN C. LINDER1 AND L. ANDREW STAEHELIN
Department of Molecular, Cellular and Developmental Biology, University of Colorado, Boulder, Colorado 80309 Received May 21, 1980 This report presents the description of a Hew organelle in a trypanosomatid flagellate. The
Leptomonas membrane lattice is a discrete organelle composed of branched and interconnected smooth membrane tubules. These 50-nm tubules frequently form an almost crystalline array. The P-fracture face of the lattice is densely covered with 7.5- to 10-nmintramembrane particles, while the E face contains very few particles. This particle distribution reflects the bilayer continuity observed between the membrane lattice and both the outer membrane of the nuclear envelope and the rough endoplasmic reticulum. The membrane lattice is also frequently associated with the Golgi apparatus and the spongiome tubules of the contractile vacuole apparatus. Although the ultrastructure of parasitic protozoa from the family T r y p a n o s o m a t i dae has been studied for m a n y years (reviewed by Vickerman and Preston, 1976), no previous report has described an organelle consisting of a complex and regular array of m e m b r a n e tubules. Such an organelle has now been found in L e p t o m o n a s coUosoma, a small nonpathogenic flagellate which normally inhabits the hindgut of the waterstrider (Wallace et al., 1960). Leptom o n a s thus appears to be unique a m o n g t r y p a n o s o m a t i d s in possessing the highly ordered elaboration of endoplasmic reticulum (ER) which we have t e r m e d the membrane lattice. T h i n section and freeze-fracture electron microscopy have enabled us to characterize these specialized membranes and to examine their relationships to other intracellular organelles.
1979). Some cells were treated with 40 mM TrisMaleate, pH 7.2, containing 0.7 mM Pb (NO~)2,4 mM MgC12, 100 mM KCI, and 20 mM NaC1 for 1.5 hr between the glutaraldehyde and osmium fixations. RESULTS
L e p t o m o n a s is a slender flagellate with a single anterior flagellum and a central nucleus (Linder, 1980; Linder and Staehelin, 1977). A prominent contractile vacuole apparatus is found adjacent to the flagellar pocket and near the Golgi apparatus (Linder and Staehelin, 1979). T h e three-dimensional m e m b r a n e lattice is a discrete b o d y formed by branched and interconnected s m o o t h m e m b r a n e tubules (Fig. 1). One lattice, or rarely two, are found in the anterior end of the cell, often near the plasma membrane. Longitudinal sections show the lattice located between the nucleus and the contractile vacuole apparatus. In m a n y instances, the lattice takes on a semicrystalMATERIALS AND METHODS line form (Figs. 2, 3), and corresponds to A culture of Leptomonas collosoma was the gift of Dr. F. G. Wallace, Department of Zoology,University w h a t mathematicians call an "infinite peof Minnesota. Cell culture methods, glutaraldehyde riodic minimal surface" (Schoen, 1970). fixationi freeze-substitution, and freeze-fracture tech- Thus, the m e m b r a n e s of the lattice form a niques using both conventionaland ultrarapid freezing continuous surface t h a t separates two conhave been described previously (Linder and Staehelin, tinuous interwoven and unlike compart~Present address: Department of Psychiatry M- ments, one being the cytoplasm occupying 003, University of California, San Diego, La Jolla, the space surrounding the tubules, and the other being the lumen within the tubules. Calif. 92093. 200 0022-5320/80/080200-06502.00/0 Copyright© 1980by AcademicPress, Inc. All rights of reproductionin any formreserved.
THE TRYPANOSOMATID MEMBRANE LATTICE T h e lattice structure, however, a p p e a r s to be quite flexible, with m a n y lattices showing v e r y little order (Fig. 1). T h e smoothsurfaced lattice tubules are usually 40-55 n m in diameter. T h e P-fracture face of the lattice is densely covered with 7.5- to 10-nm i n t r a m e m b r a n e particles while the complem e n t a r y E face contains v e r y few particles (Figs. 4, 5). T h e surface m e m b r a n e s of the lattice structure are frequently continuous with cisternae of the rough E R (Fig. 1). I n fact, a large a r e a of the lattice is usually b o u n d e d b y rough E R which at m a n y points loses the ribosomes f r o m the lattice-facing surface and evaginates to f o r m the lattice tubules (Figs. 1, 4, 5). Alternatively, the lattice m a y exhibit a similar relationship with the outer m e m b r a n e of the nuclear envelope (Figs. 4, 6). T h e particle distribution on the lattice m e m b r a n e faces clearly r e s e m b l e s t h a t of the E R and the nuclear envelope. One or several Golgi complexes are often found nearby, s o m e t i m e s closely a p p r e s s e d to the lattice (Figs. 5, 7). Frequently, vesicles a p p e a r to arise f r o m the b o u n d a r y of the m e m b r a n e lattice adjacent to the forming face of a Golgi stack, just as t h e y do f r o m n o r m a l E R cisternae. Although a n edge of the lattice not b o u n d e d b y rough E R is usually in v e r y close proximity to the spiny-coated spongiome tubules of the contractile vacuole a p p a r a t u s (Linder a n d Staehelin, 1979), we h a v e not b e e n able to establish unequivocally t h a t a direct bilayer
201
continuity exists b e t w e e n the two t y p e s of m e m b r a n e (Figs. 1, 6). W h e n whole cells are i n c u b a t e d in a lead-containing buffer, the m e m b r a n e s of the lattice, endoplasmic reticulum, a n d nuclear envelope selectively bind the lead precipitate, while the spongiome tubules, Golgi apparatus, and contractile vacuole r e m a i n unstained (Fig. 7). T h e m e m b r a n e lattice is not a constant c o m p o n e n t of Leptomonas cells. I t s presence a p p e a r s to be d e p e n d e n t on environm e n t a l conditions. Lattices are either found in all cells of a culture or in none of them. I n cultures grown u n d e r s t a n d a r d conditions or t r e a t e d with the n o r m a l 0.16 M p h o s p h a t e buffer, lattices were a b s e n t f r o m all cells in only 21 of 53 e x p e r i m e n t s (39.6%). I n contrast, cells placed in diluted m e d i u m or low osmolarity buffer (40 m M E D T A , 50 m M Tris-HC1) for times of 1 hr or less lacked lattices in 20 out of 26 e x p e r i m e n t s (76.9%). T h e fixation conditions did not s e e m relevant, because lattices were found in cells p r e p a r e d for n o r m a l thin sectioning, freeze-substituted, frozen with cryoprotectants, or ultrarapidly frozen. DISCUSSION Although the E R of Leptomonas is associated with the peripheral microtubules, nucleus, and Golgi a p p a r a t u s as in o t h e r t r y p a n o s o m a t i d s (Linder, 1980), its elaboration into a complex lattice of m e m b r a n e tubules is something novel. A b u n d a n t s m o o t h m e m b r a n e tubules h a v e b e e n seen
FIG. 1. Thin section of a membrane lattice near the flagellar pocket (P). It is associated with the contractile vacuole (CV) and the tubules of the spongiome (S). Note the boundary of rough ER at one edge (arrow). × 43 200. Fro. 2. Thin section of a highly ordered membrane lattice which is composed of 40- to 55-rim smoothsurfaced tubules. × 82 000. Fro. 3. Freeze-fracture micrograph of a highly ordered membrane lattice. × 79 700. FIG. 4. The E-fracture face (EF) at the surface of a membrane lattice resembles that of the nuclear envelope (N) to which it is connected (arrowheads). :Both have very few particles. × 58 200. FIG. 5. The lattice P face (PF), in contrast, contains many 7.5- to 10-nm particles, similar to the P face of the nuclear envelope. The boundary of this lattice has invaginated to form tubules only in certain regions, leaving large areas which resemble undifferentiated endoplasmic reticulum. Golgi apparatus (G). × 58 200. FIG. 6. The outer membrane of the nuclear envelope is continuous with the lattice membrane in this thin section (arrowheads). Spongiome tubules (S) are found very close to the membrane lattice (ML). Nucleus (N). × 67 400.
202
LINDER AND STAEHELIN
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LINDER AND STAEHELIN
FIG. 7. The membranes of the lattice (ML), nucleus (N), and endoplasmicreticulum (ER) specificallybind a lead precipitate after whole cells are incubated in a lead-containingbuffer.The Golgimembranes (G) and the plasma membrane are not labeled. × 50 400. before in trypanosomatids (Brooker, 1971; Steiger, 1973; Vickerman, 1969), but never arranged as a highly ordered, discret organelle like the Leptomonas membrane lattice. Although the specific chemical reaction producing the lead binding is not known, these results and the distinctive intramembrane particle distribution both support the idea that the membranes of the lattice are basically similar to those of the ER and the nuclear envelope, with which they are continuous. The function of the membrane lattice is at this time unknown, but similarly elaborated smooth membranes have previously been found in a wide variety of cells. Their roles have usually been mysterious, although frequent suggestions include either
membrane storage, as in the prolamellar body of dark-grown chloroplasts (Gunning, 1965), or else secretion and excretion. This latter purpose has been suggested for the complex membranes found in a fish gland (Lennep and Lanzing, 1967), plant nectaries (Eym6, 1966), and in the fungus Ascobolus (Zachariah and Anderson, 1973). This type of membrane thus appears to be a specialization of the endoplasmic reticulum whose lattice-like structure could provide either a compact packing of stored membrane material or else a large surface area in contact with the cytoplasm, proving significant for transport activities. The development of a lattice in Leptomonas may be dependent on some component of the ionic or growth conditions. Its variability with the osmolar-
THE TRYPANOSOMATID MEMBRANE LATTICE ity of t h e e n v i r o n m e n t suggests a f u n c t i o n a l l i n k to t h e c o n t r a c t i l e v a c u o l e m e m b r a n e s , a l t h o u g h t h a t r e m a i n s to b e i n v e s t i g a t e d . This research was supported by National Institutes of Health Grant GM18639 to L. A. Staehelin and National Science Foundation predoctoral fellowship to J. C. Linder. REFERENCES BROOKER, B. E. (1971) Z. Zellforsch. Mikrosk. Anat. 105, 155-166. EYMI~, J. (1966) C. R. Acad. Sci. (Paris) Ser. D 262, 1629-1632. GUNNING,B. E. S. (1965} Protoplasma 60, 111-130. LENNEP, E. W. VAN, AND LANZING, W. J. R. (1967) J. Ultrastruct. Res. 18, 333-344.
205
LINDER, J. C. (1980) Ph.D. thesis, University of Colorado, Boulder, Colo. LINDER, J. C., AND STAEHELIN,L. A. (1977) J. Ultrastruct. Res. 60, 246-262. LINDER, J. C., AND STAEHELIN, L. A. (1979) J. Cell Biol. 83, 371-382. SCHOEN, A. H. (1970) NASA Technical Note TN-D5541; C98, 1-92. STEIGER, R. F. (1973) Acta Trop. 30, 64-168. VICKERMAN,K. (1969) J. Protozool. 16, 54-69. VICKERMAN, K., AND PRESTON, T. M. (1976) In LUMSDEN, W. H. R., AND EVANS, D. A. (Eds.),
Biologyof
the Kinetoplastida, Vol. 1, pp. 35-130, Academic Press, New York. WALLACE, F. G., CLARK, T. B., DYER, M. I., AND COLLINS,T. (1960) J. Protozool. 7, 390-392. ZACHARIAH, K., AND ANDERSON, R. H. (1973) J. Ultrastruct. Res. 44, 405-420.
72, 2 0 0 - 2 0 5
(1980)
The Membrane Lattice: A Novel Organelle of the Trypanosomatid Flagellate Leptomonas collosoma JEAN C. LINDER1 AND L. ANDREW STAEHELIN
Department of Molecular, Cellular and Developmental Biology, University of Colorado, Boulder, Colorado 80309 Received May 21, 1980 This report presents the description of a Hew organelle in a trypanosomatid flagellate. The
Leptomonas membrane lattice is a discrete organelle composed of branched and interconnected smooth membrane tubules. These 50-nm tubules frequently form an almost crystalline array. The P-fracture face of the lattice is densely covered with 7.5- to 10-nmintramembrane particles, while the E face contains very few particles. This particle distribution reflects the bilayer continuity observed between the membrane lattice and both the outer membrane of the nuclear envelope and the rough endoplasmic reticulum. The membrane lattice is also frequently associated with the Golgi apparatus and the spongiome tubules of the contractile vacuole apparatus. Although the ultrastructure of parasitic protozoa from the family T r y p a n o s o m a t i dae has been studied for m a n y years (reviewed by Vickerman and Preston, 1976), no previous report has described an organelle consisting of a complex and regular array of m e m b r a n e tubules. Such an organelle has now been found in L e p t o m o n a s coUosoma, a small nonpathogenic flagellate which normally inhabits the hindgut of the waterstrider (Wallace et al., 1960). Leptom o n a s thus appears to be unique a m o n g t r y p a n o s o m a t i d s in possessing the highly ordered elaboration of endoplasmic reticulum (ER) which we have t e r m e d the membrane lattice. T h i n section and freeze-fracture electron microscopy have enabled us to characterize these specialized membranes and to examine their relationships to other intracellular organelles.
1979). Some cells were treated with 40 mM TrisMaleate, pH 7.2, containing 0.7 mM Pb (NO~)2,4 mM MgC12, 100 mM KCI, and 20 mM NaC1 for 1.5 hr between the glutaraldehyde and osmium fixations. RESULTS
L e p t o m o n a s is a slender flagellate with a single anterior flagellum and a central nucleus (Linder, 1980; Linder and Staehelin, 1977). A prominent contractile vacuole apparatus is found adjacent to the flagellar pocket and near the Golgi apparatus (Linder and Staehelin, 1979). T h e three-dimensional m e m b r a n e lattice is a discrete b o d y formed by branched and interconnected s m o o t h m e m b r a n e tubules (Fig. 1). One lattice, or rarely two, are found in the anterior end of the cell, often near the plasma membrane. Longitudinal sections show the lattice located between the nucleus and the contractile vacuole apparatus. In m a n y instances, the lattice takes on a semicrystalMATERIALS AND METHODS line form (Figs. 2, 3), and corresponds to A culture of Leptomonas collosoma was the gift of Dr. F. G. Wallace, Department of Zoology,University w h a t mathematicians call an "infinite peof Minnesota. Cell culture methods, glutaraldehyde riodic minimal surface" (Schoen, 1970). fixationi freeze-substitution, and freeze-fracture tech- Thus, the m e m b r a n e s of the lattice form a niques using both conventionaland ultrarapid freezing continuous surface t h a t separates two conhave been described previously (Linder and Staehelin, tinuous interwoven and unlike compart~Present address: Department of Psychiatry M- ments, one being the cytoplasm occupying 003, University of California, San Diego, La Jolla, the space surrounding the tubules, and the other being the lumen within the tubules. Calif. 92093. 200 0022-5320/80/080200-06502.00/0 Copyright© 1980by AcademicPress, Inc. All rights of reproductionin any formreserved.
THE TRYPANOSOMATID MEMBRANE LATTICE T h e lattice structure, however, a p p e a r s to be quite flexible, with m a n y lattices showing v e r y little order (Fig. 1). T h e smoothsurfaced lattice tubules are usually 40-55 n m in diameter. T h e P-fracture face of the lattice is densely covered with 7.5- to 10-nm i n t r a m e m b r a n e particles while the complem e n t a r y E face contains v e r y few particles (Figs. 4, 5). T h e surface m e m b r a n e s of the lattice structure are frequently continuous with cisternae of the rough E R (Fig. 1). I n fact, a large a r e a of the lattice is usually b o u n d e d b y rough E R which at m a n y points loses the ribosomes f r o m the lattice-facing surface and evaginates to f o r m the lattice tubules (Figs. 1, 4, 5). Alternatively, the lattice m a y exhibit a similar relationship with the outer m e m b r a n e of the nuclear envelope (Figs. 4, 6). T h e particle distribution on the lattice m e m b r a n e faces clearly r e s e m b l e s t h a t of the E R and the nuclear envelope. One or several Golgi complexes are often found nearby, s o m e t i m e s closely a p p r e s s e d to the lattice (Figs. 5, 7). Frequently, vesicles a p p e a r to arise f r o m the b o u n d a r y of the m e m b r a n e lattice adjacent to the forming face of a Golgi stack, just as t h e y do f r o m n o r m a l E R cisternae. Although a n edge of the lattice not b o u n d e d b y rough E R is usually in v e r y close proximity to the spiny-coated spongiome tubules of the contractile vacuole a p p a r a t u s (Linder a n d Staehelin, 1979), we h a v e not b e e n able to establish unequivocally t h a t a direct bilayer
201
continuity exists b e t w e e n the two t y p e s of m e m b r a n e (Figs. 1, 6). W h e n whole cells are i n c u b a t e d in a lead-containing buffer, the m e m b r a n e s of the lattice, endoplasmic reticulum, a n d nuclear envelope selectively bind the lead precipitate, while the spongiome tubules, Golgi apparatus, and contractile vacuole r e m a i n unstained (Fig. 7). T h e m e m b r a n e lattice is not a constant c o m p o n e n t of Leptomonas cells. I t s presence a p p e a r s to be d e p e n d e n t on environm e n t a l conditions. Lattices are either found in all cells of a culture or in none of them. I n cultures grown u n d e r s t a n d a r d conditions or t r e a t e d with the n o r m a l 0.16 M p h o s p h a t e buffer, lattices were a b s e n t f r o m all cells in only 21 of 53 e x p e r i m e n t s (39.6%). I n contrast, cells placed in diluted m e d i u m or low osmolarity buffer (40 m M E D T A , 50 m M Tris-HC1) for times of 1 hr or less lacked lattices in 20 out of 26 e x p e r i m e n t s (76.9%). T h e fixation conditions did not s e e m relevant, because lattices were found in cells p r e p a r e d for n o r m a l thin sectioning, freeze-substituted, frozen with cryoprotectants, or ultrarapidly frozen. DISCUSSION Although the E R of Leptomonas is associated with the peripheral microtubules, nucleus, and Golgi a p p a r a t u s as in o t h e r t r y p a n o s o m a t i d s (Linder, 1980), its elaboration into a complex lattice of m e m b r a n e tubules is something novel. A b u n d a n t s m o o t h m e m b r a n e tubules h a v e b e e n seen
FIG. 1. Thin section of a membrane lattice near the flagellar pocket (P). It is associated with the contractile vacuole (CV) and the tubules of the spongiome (S). Note the boundary of rough ER at one edge (arrow). × 43 200. Fro. 2. Thin section of a highly ordered membrane lattice which is composed of 40- to 55-rim smoothsurfaced tubules. × 82 000. Fro. 3. Freeze-fracture micrograph of a highly ordered membrane lattice. × 79 700. FIG. 4. The E-fracture face (EF) at the surface of a membrane lattice resembles that of the nuclear envelope (N) to which it is connected (arrowheads). :Both have very few particles. × 58 200. FIG. 5. The lattice P face (PF), in contrast, contains many 7.5- to 10-nm particles, similar to the P face of the nuclear envelope. The boundary of this lattice has invaginated to form tubules only in certain regions, leaving large areas which resemble undifferentiated endoplasmic reticulum. Golgi apparatus (G). × 58 200. FIG. 6. The outer membrane of the nuclear envelope is continuous with the lattice membrane in this thin section (arrowheads). Spongiome tubules (S) are found very close to the membrane lattice (ML). Nucleus (N). × 67 400.
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203
204
LINDER AND STAEHELIN
FIG. 7. The membranes of the lattice (ML), nucleus (N), and endoplasmicreticulum (ER) specificallybind a lead precipitate after whole cells are incubated in a lead-containingbuffer.The Golgimembranes (G) and the plasma membrane are not labeled. × 50 400. before in trypanosomatids (Brooker, 1971; Steiger, 1973; Vickerman, 1969), but never arranged as a highly ordered, discret organelle like the Leptomonas membrane lattice. Although the specific chemical reaction producing the lead binding is not known, these results and the distinctive intramembrane particle distribution both support the idea that the membranes of the lattice are basically similar to those of the ER and the nuclear envelope, with which they are continuous. The function of the membrane lattice is at this time unknown, but similarly elaborated smooth membranes have previously been found in a wide variety of cells. Their roles have usually been mysterious, although frequent suggestions include either
membrane storage, as in the prolamellar body of dark-grown chloroplasts (Gunning, 1965), or else secretion and excretion. This latter purpose has been suggested for the complex membranes found in a fish gland (Lennep and Lanzing, 1967), plant nectaries (Eym6, 1966), and in the fungus Ascobolus (Zachariah and Anderson, 1973). This type of membrane thus appears to be a specialization of the endoplasmic reticulum whose lattice-like structure could provide either a compact packing of stored membrane material or else a large surface area in contact with the cytoplasm, proving significant for transport activities. The development of a lattice in Leptomonas may be dependent on some component of the ionic or growth conditions. Its variability with the osmolar-
THE TRYPANOSOMATID MEMBRANE LATTICE ity of t h e e n v i r o n m e n t suggests a f u n c t i o n a l l i n k to t h e c o n t r a c t i l e v a c u o l e m e m b r a n e s , a l t h o u g h t h a t r e m a i n s to b e i n v e s t i g a t e d . This research was supported by National Institutes of Health Grant GM18639 to L. A. Staehelin and National Science Foundation predoctoral fellowship to J. C. Linder. REFERENCES BROOKER, B. E. (1971) Z. Zellforsch. Mikrosk. Anat. 105, 155-166. EYMI~, J. (1966) C. R. Acad. Sci. (Paris) Ser. D 262, 1629-1632. GUNNING,B. E. S. (1965} Protoplasma 60, 111-130. LENNEP, E. W. VAN, AND LANZING, W. J. R. (1967) J. Ultrastruct. Res. 18, 333-344.
205
LINDER, J. C. (1980) Ph.D. thesis, University of Colorado, Boulder, Colo. LINDER, J. C., AND STAEHELIN,L. A. (1977) J. Ultrastruct. Res. 60, 246-262. LINDER, J. C., AND STAEHELIN, L. A. (1979) J. Cell Biol. 83, 371-382. SCHOEN, A. H. (1970) NASA Technical Note TN-D5541; C98, 1-92. STEIGER, R. F. (1973) Acta Trop. 30, 64-168. VICKERMAN,K. (1969) J. Protozool. 16, 54-69. VICKERMAN, K., AND PRESTON, T. M. (1976) In LUMSDEN, W. H. R., AND EVANS, D. A. (Eds.),
Biologyof
the Kinetoplastida, Vol. 1, pp. 35-130, Academic Press, New York. WALLACE, F. G., CLARK, T. B., DYER, M. I., AND COLLINS,T. (1960) J. Protozool. 7, 390-392. ZACHARIAH, K., AND ANDERSON, R. H. (1973) J. Ultrastruct. Res. 44, 405-420.