Download a biological system producing a self-assembling

J. Cell Set. 8, 93-109(1971)
93
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A BIOLOGICAL SYSTEM PRODUCING A
SELF-ASSEMBLING CHOLESTERIC PROTEIN
LIQUID CRYSTAL
A. C. NEVILLE AND B. M. LUKE
A.R.C. Unit of Insect Physiology, Department of Zoology,
South Parks Road, Oxford, England
SUMMARY
The protein in the oothecal glands of praying mantids (Sphodromantis tenuidentata, Miomantis monacha) exists in the form of lamellar liquid crystalline spherulites, which coalesce as
theyflowout of a punctured gland tubule. Electron micrographs of sections of these spherulites
after fixation show parabolic patterns of an electron-light component, set in a continuous matrix
of protein. Such patterns arise in helicoidal systems (e.g. arthropod cuticle) and microdensitometric scans of the matrix show a rhythmical electron-density variation consistent with helicoidal structure. Double spiral patterns identical to those seen in liquid crystal spherulites are
illustrated. These properties resemble those of cholesteric liquid crystals. The constructional
units appear to be molecular rather than fibrillar as described by previous authors. The
helicoidal architecture arises by self-assembly in the gland lumen. Lamellar surface structures
self-assembled spontaneously on glass coverslips when the protein was left to stand for several
days. When heated to 55 °C, the birefringent liquid crystalline protein abruptly changes to an
isotropic gel, with associated loss of parabolic patterning in electron micrographs and of the
rhythmical electron-density variation on microdensitometric scans. This behaviour is compared
to the formation of gelatin from collagen, in terms of the randomization of an originally ordered
secondary structure.
INTRODUCTION
Previous studies on mantid oothecal protein have shown that it is an a-protein,
which can be converted into ribbons by treatment in vitro with calcium ions (Rudall,
1956); also, within the colleterial glands it exists in spherulites, suitable sections of
which show a double birefringent spiral (Kenchington & Flower, 1969), like those
seen in cholesteric liquid crystal spherulites (Robinson, 1966; Wilkins, 1963). Electron
micrographs of thin sections of such spherulites showed a parabolic pattern (Kenchington & Flower, 1969), which can be derived from oblique sections through a
helicoidal structure (Bouligand, 1965). (The basis of helicoidal structure is shown in
Fig. 1, p. 95). Our own work, which has proceeded independently from that of
Kenchington & Flower (1969) but which was inspired by a polarized-light micrograph of a double spiral in oothecal protein from Sphodromantis centralis (Kenchington, 1965), supports the helicoidal interpretation of the protein spherulites; but our
interpretation of the units which form such helicoids differs radically from that of the
above workers. Furthermore, we wish to demonstrate the self-assembly of such
helicoids, and to emphasize their liquid crystalline nature.
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A. C. Neville and B. M. Luke
MATERIAL AND METHODS
Protein samples were obtained from the left colleterial gland of adult female Sphodromantis
tenuidentata, reared from eggs in the laboratory by Mr C. W. Berg. Results were confirmed on
a South African mantis, Miomantis monacha. Material for electron microscopy was prepared
by fixing whole gland tubules for 2 h in 2-5 % glutaraldehyde in 0-05 M cacodylate buffer at
pH 7-2 at 4 CC. It was washed in several changes of buffer at 4 °C for 0-5 h, then immersed in
1 % aqueous osmium tetroxide for 1 h. The tubules were transferred directly to 70 % ethanol,
dehydrated, transferred to propylene oxide, and embedded in Araldite. Thin sections were
stained either in a saturated solution of uranyl acetate in 50 % ethanol, or in 2 % aqueous
potassium permanganate, followed in both cases by lead citrate. Sections were examined in an
AEI EM 6 B electron microscope. Microdensitometric scans of electron micrographs were made
on a Joyce double-beam automatic recording microdensitometer. Polarization and phasecontrast microscopy were carried out with Zeiss polarizing and phase-contrast microscopes,
and observations at controlled temperature were made using a Kofler hot microscope stage.
RESULTS
Spherulite ultrastructure
Fixed material was sectioned in situ in the oothecal gland. The system appears
lamellar with a periodicity of 1-3 /tm, and double spirals of lamellae were frequently
seen both in phase contrast (Fig. 3) and in the electron microscope (Fig. 4).
Observation of unfixed material in freshly killed mantids shows that the oothecal
gland is packed with lamellar spherulites best observed between crossed polaroids
(Fig. 5). A typical isolated spherulite is photographed between crossed polaroids in
Fig. 6. The distortion from truly spherical shape was typical in our material. Use of
a 550-nm retardation plate shows the orientation of construction units to be circumferential, birefringence being positive parallel to the lamellae. The single nature of the
extinction rings (lamellae) shows the system to be uniaxial, as distinct from biaxial
(double rings). The spherulites were stable for many weeks when mounted in
glycerol. Electron micrographs (Fig. 14) show that the edges of spherulites appear
'sticky', with strands of material perhaps forming by contact with neighbouring
spherulites. Rudall (1956) described the globules, which he showed to be protein, as
corpuscles moving in the gland serum. We confirm this observation, but wish to
extend it by distinguishing between the corpuscular mobility and the actual flow of
the protein itself; the difference, which is an important one, is easily seen with a
polarizing microscope. A fresh colleterial gland tubule was punctured, and the
spherulites seen to coalesce as theyflowedout through the puncture, forming a system
with preferred orientation in the direction of flow. This system coiled backwards and
forwards upon itself some distance outside the tubule. These observations show that
the protein in the gland is in a liquid crystalline state.
The construction units of spherulites have been described by Rudall (1956) as
fibrils 0-2-0-5 /tm in diameter, and by Kenchington & Flower (1969) as fibrils
0-05-0-1 /tm in diameter. Such fibrils were never seen in any of oui electron micrographs. On the contrary, the oothecal protein forms a continuous phase which can be
traced at high magnification (x 250000) with no discontinuities throughout a section,
and which appears electron-dense after staining with potassium permanganate. The
Protein liquid crystal from a biological system
95
less electron-dense discontinuous phase seen in the micrographs (Figs. 4, 8, 9) has
not yet been identified. It is probably pushed into a helicoidal array by the surrounding
continuous phase protein. A comparable system is the moulding of pore canals into
twisted ribbons by the crystalline helicoidal array of microfibrils in insect cuticles
(Neville & Luke, 1969a).
Evidence for helicoidal structure
The electron-light component forms a useful natural marker system, displaying the
parabolic patterns typical of a helicoidal system (Fig. 8). Such patterns have been
analysed for crustacean cuticle microfibrils (Bouligand, 1965), and for insect cuticle
pore canals (Neville, Thomas & Zelazny, 1969).
Lamellar period
|\
A
\
Helicoidal pitch
Fig. 1. Diagram illustrating the principle of helicoidal structure. Representative
planes (drawn as circles) of anisometric and parallel construction units are drawn for
every 45 ° of rotation about the axis running through the centres of all the circles.
Orientation of the units is shown by arrows. Between crossed polaroids the units which
lie in the plane of the page would show maximal birefringence, appearing as lamellae.
Hence the lamellar period is half the true pitch of the helicoid.
The electron-density variation across such micrographs as Fig. 8 was measured with
a microdensitometer. The rhythmical variation (Fig. 2 A) is consistent with the steady
rotation (theoretically sinusoidal) of the units in a helicoidal structure as in Fig. 1.
Superimposed upon this are seen the troughs caused by the electron-light discontinuous phase (arrows in Fig. 2 A). The presence of a double spiral at the centre of a
spherulite (Fig. 4) also supports a helicoidal explanation for this structure by comparison with light-microscopical observations on cholesteric liquid crystals, and is
discussed below. The evidence supports the hypothesis for the helicoidal nature of
cholesteric liquid crystals.
Evidence for self-assembly
Our electron micrographs show that helicoidal structure arises extracellularly
(Fig. 11), no parabolic patterns appearing in the cells. Extensive areas of parabolic
patterning are restricted to the lumen of the gland, occurring at a distance of 3 /tm
from the apical border of the gland cells and 1 /tm from the luminal cells in which the
A. C. Neville and B. M. Luke
9.6
gland cells are embedded. The intervening space is filled with the so-called serum
(Fig. 13). The amount of secretion present within the end-apparatus of the gland cells
is insufficient to enable us to determine whether it is already helicoidal. Since there
are no discontinuities over extensive volumes of the final spherulites, the products
extruding from the gland cells must be capable of assembling on to the material
which has already been secreted. Thus the formation of helicoidal architecture occurs
by an extracellular self-assembly process in the gland lumen.
180°
360°
1 /im
Fig. 2. Microdensitometric scans of electron-density variation across electron-microscope plates of (A) helicoidal mantis oothecal protein like that shown in Fig. 8, and
(B) the same after gel formation at 55 °C, as shown in Fig. 9. The density variation seen
in A varies with lamellar periodicity, reflecting the helicoidal rotation of component
units in the densely stained matrix. This variation had disappeared in B, but the
electron-light component (arrows) is still present. Angle of rotation of helicoid is
indicated in A, with 0° and 3600 lying in the plane of the page.
Lamellar surface structures formed in vitro by self-assembly on a glass coverslip
or on the surface of a glass tube, when oothecal protein was left in locust saline for
several days. These surface structures (Fig. 7) closely resemble those formed on glass
surfaces by solutions of poly-y-benzyl D-glutamate and poly-y-benzyl L-glutamate in
dioxan (Robinson, 1958). They provide further evidence for the self-assembling
properties of mantis oothecal protein.
Gel formation
When extracted oothecal protein is heated on a microscope slide with a Kofler hot
stage, during continuous observation between crossed polaroids, a dramatic change is
seen at a critical temperature of 55 °C. The previously flowing and birefringent liquid
crystalline phase abruptly changes into a static and isotropic gel. Lowering the
temperature showed the change to be irreversible. The system has changed in physical
state from a liquid crystal to a hydrated rubber-like gel of low tensile strength, which
develops cracks on deformation, and shows reversible strain birefringence when
Protein liquid crystal from a biological system
97
stresses lower than that causing tensile failure are applied. Identical results were
obtained with protein from both species of mantid tested.
The above procedure was repeated and the resulting gel fixed for electron microscopy. Thin sections showed that the helicoidal pattern had disappeared, leaving a
random matrix with the electron-light discontinuous phase still present (Fig. 9).
(Gelling prior to fixation resulted in harder material causing the scratches in Fig. 9.)
Microdensitometiic scanning confirmed the abolition of the rhythmical variation in
electron density, but the electron-light component was still represented by troughs
(arrows, Fig. 2B).
Cytology
The general histological appearance of the secretory cells at the electron-microscope
level has been described for the closely similar cockroach left colleterial gland in a
pioneer paper on insect gland cell ultrastructure (Mercer & Brunet, 1959). The
equivalent details of the mantid left colleterial gland have been given by Kenchington
& Flower (1969). Whilst confirming the fundamentals of these descriptions, we wish
to add the following details.
Gland cells. Prior to secretion, the oothecal protein occurs as vesicles in the cells
(Fig. 12), which, by contrast with normal epidermal cells secreting cuticle, are very
rich in rough endoplasmic reticulum, suggesting that the protein is synthesized for
export in the gland cells themselves. (We note this feature because epidermal cells in
general may obtain some of the proteins which they subsequently secrete from elsewhere in the body via the haemocoel. This may be deduced from the electrophoresis
results of Fox & Mills, 1969.) The microvillate end-apparatus through which secretion of the mantis oothecal protein occurs is typical of insect gland cells in general
(Mercer & Brunet, 1959; Gupta & Smith, 1969). As in the colleterial glands of
Saturniid moths (Berry, 1968), several of the mantid colleterial gland cells contain
cytolysomes with myelin-like figures.
Lumen cells. Colleterial gland cells are set in an epithelium otherwise composed of
so-called ' chitinogenous' cells. Whilst agreeing with this general layout, we disagree
with previous workers (Mercer & Brunet, 1959; Kenchington & Flower, 1969) in the
naming of these cells. The structure bordering the lumen of the organ resembles an
epicuticle in ultrastructure and thickness (Figs. 10, 11, 13). Since epicuticle does not
contain chitin, we therefore propose to call the cells responsible for this structure
'lumen cells'. In the mantids they contain numerous microtubules oriented parallel
to the surface of the lumen. This has previously been noted by Berry (1968) in
Saturniid moth colleterial glands.
DISCUSSION
Helicoidal ultrastructure
The above ultrastructural evidence supports the theory, based upon optical properties, of the helicoidal structure of cholesteric liquid crystals (Friedel, 1922). The
mantis oothecal protein appears potentially useful for building chemical and archi7
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tectural models of cuticle, and for experiments on helicoid self-assembly. It emerges
that protein can form a helicoid in the absence of chitin (Hackman & Goldberg,
i960, have shown that chitin is absent from the oothecal protein of a mantid, Orthodera
ministralis), but this does not necessarily imply that protein is the prime factor
governing assembly of helicoids in arthropod cuticle.
We have suggested that the helicoidal structure of arthropod cuticle in general
might arise by subsequent stabilization of a self-assembling cholesteric liquid crystalline deposition zone present as a thin region next to the cuticle-secreting epidermal
cells (Neville & Luke, 19690,6; Neville & Caveney, 1969). Electron-microscope
images show evidence of helicoidal structure in this deposition zone. The fact that
we have shown above that it is possible to fix and visualize a cholesteric liquid crystalline system in the electron microscope, does not detract from the hypothesis.
Significance of the double spiral pattern
Kenchington & Flower (1969) briefly mention the similarity between the double
spiral seen in light-microscope preparations of mantis spherulites, with that of
polypeptide spherulites (Robinson, 1966). We wish to extend the comparison to
include the double spiral patterns in transfer RNA spherulites (Wilkins, 1963), and
to stress that such spiral patterns arise because of geometrical reasons. The mathematical derivation of double spirals in helicoidal spherulites is discussed by Pryce &
Frank in Robinson, Ward & Beevers (1958). They show that sections through a
helicoidal spherulite will always contain a double spiral pattern except in the plane
of the single radial line of disinclination, which is also a geometrical consequence of
their construction. Double spirals have also been seen in sections of tubercles in crab
cuticle and their origin is beautifully demonstrated in diagrams by Bouligand (1965).
They also occur in sections of corneal lenses of some arthropod eyes (Horridge, 1969;
S. Caveney, unpublished), where they arise from a hemisphere of cuticle with
helicoidal construction.
Spherulite construction units
With regard to the units from which the helicoids are built, we disagree with the
interpretation of Kenchington & Flower (1969). They described the units as electrondensely staining 'fibrils' arranged at angles of 18 ° to each other, for which they have
specifically constructed a perspex model. By contrast, we regard the construction
units as unresolvable by electron microscopy of thin sections. A more likely candidate
for a unit could be the twin-coiled a-helices postulated by Rudall (1956) on the
basis of X-ray diffraction of artificial fibres pulled out from the viscous protein in the
gland. It is significant that the building units of other helicoidal systems are also
asymmetrical (cholesteryl derivatives), often with helical components (transfer RNA)
or even totally helical (synthetic polypeptides and DNA: Robinson, 1961).
We find no discontinuities in the electron densely-staining matrix, and this is
supported by the gelling experiments in which a continuous isotropic gel was formed.
Protein liquid crystal from a biological system
99
One common feature of all the helicoidal systems we have worked with is that they
are capable of showing a very wide range of pitch (see Fig. 1, p. 95), reflecting a wide
variation of angle between successive planes of units.
Gel formation
The formation of a gel from mantis oothecal protein clearly differs from the
shrinkage of collagen, since the latter phenomenon is readily reversible. A closer
analogy is the formation of gelatin from collagen (Harkness, 1961), which involves
the irreversible denaturation of the secondary structure, and the formation of a new
random configuration with higher entropy. We appear to have converted an ordered
liquid crystal into a random gel, perhaps by the breakdown of the H-bonds in the
secondary structure of the original units, followed by the formation of a new random
secondary structure. The breaking of the original H-bonding (for instance in the
twin a-helices postulated by Rudall, 1956), could explain the dramatically sudden
disappearance of the helicoidal architecture.
We wish to thank Mr C. W. Berg for rearing the mantids and Mr S. Caveney for obtaining
Miomantis monacha. We thank Prof. J. W. S. Pringle, F.R.S., for his comments on the manuscript. Finally, our grateful thanks to the Agricultural Research Council for full financial
support.
REFERENCES
BERRY, S. J. (1968). The fine structure of the colleterial glands of Hyalophora cecropia (Lepidoptera). J. Morpli. 125, 259-280.
BOULIGAND, Y. (1965). Sur une architecture torsadde r^pandue dans de nombreuses cuticules
d'arthropodes. C. r. hebd. Se'anc. Acad. Sci., Paris 261, 3665-3668.
Fox, F. R. & MILLS, R. R. (1969). Changes in haemolymph and cuticle proteins during the
moulting process in the American cockroach. Comp. Biochem. Physiol. 29, 1187-1195.
FRIEDEL, M. G. (1922). Les etats mesomorphes de la matiere. Annls Phys. 18, 273-474.
GUPTA, B. L. & SMITH, D. S. (1969). Fine structural organization of the spermatheca in the
cockroach, Periplaneta americana. Tissue & Cell 1, 295-324.
HACKMAN, R. H. & GOLDBERG, M. (i960). Composition of the oothecae of three Orthoptera.
J. Insect Physiol. 5, 73-78.
HARKNESS, R. D. (1961). Biological functions of collagen. Biol. Rev. 36, 399-463.
HORRIDGE, G. A. (1969). The eye of the firefly, Photuris. Proc. R. Soc. B 171, 445-463.
KENCHINCTON, W. (1965). The Structure and Function of Protein Secreting and Associated
Glands in Insects. Ph.D. Thesis, University of Leeds.
KENCHINCTON, W. & FLOWER, N. E. (1969). Studies on insect fibrous proteins: the structural
protein of the ootheca in the praying mantis, Sphodromantis centralis Rehn. J. Microscopy
89, 263-281.
MERCER, E. H. & BRUNET, P. C. J. (1959). The electron microscopy of the left colleterial gland
of the cockroach. J. biophys. biochem. Cytol. 5, 257-262.
NEVILLE, A. C. & CAVENEY, S. (1969). Scarabaeid beetle exocuticle as an optical analogue of
cholesteric liquid crystals. Biol. Rev. 44, 531-562.
NEVILLE, A. C. & LUKE, B. M. (1969a). Molecular architecture of adult locust cuticle at the
electron microscope level. Tissue & Cell 1, 355-366.
NEVILLE, A. C. & LUKE, B. M. (19696). A two-system model for chitin-protein complexes in
insect cuticles. Tissue & Cell 1, 689-707.
NEVILLE, A. C , THOMAS, M. G. & ZELAZNY, B. (1969). Pore canal shape related to molecular
architecture of arthropod cuticle. Tissue & Cell 1, 183-200.
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M. H. L. & FRANK, F. C. (1958). The spherulitic texture. Appendix to Robinson, C ,
Ward, J. C. & Beevers, R. B. (1958). Liquid crystalline structure in polypeptide solutions.
Discuss. Faraday Soc. 25, 29-42.
ROBINSON, C. (1958). Surface structures in liquid crystals. In Surface Phenomena in Chemistry
and Biology. London: Pergamon Press.
ROBINSON, C. (1961). Liquid-crystalline structures in polypeptide solutions. Tetrahedron
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RUDALL, K. M. (1956). Protein ribbons and sheets. Led. scient. Basis Med. 5, 217-230.
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{Received id June 1970)
PRYCE,
Fig. 3. Phase-contrast micrograph of i-/tm section through oothecal protein fixed
in situ in the colleterial gland of M. monacha. The lamellation is due to helicoidal
structure.
Fig. 4. Electron micrograph through centre of liquid crystalline spherulite of oothecal
gland protein of S. tenuidentata, fixed in situ in colleterial gland. The lamellae,
between which runs a parabolic pattern of obliquely sectioned electron-light components, themselves appear to coil round in a double spiral typical of the centre of a
helicoidal spherulite. x 6500.
Protein liquid crystal from a biological system
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A. C. Neville and B. M. Luke
Fig. 5. Photomicrograph of spherulites of M. monacha oothecal protein in situ
between crossed polaroids.
Fig. 6. As for Fig. 5 but a single spherulite extracted.
Fig. 7. Phase-contrast micrograph of M. monacha oothecal protein surface structures
which have reassembled on a glass surface in vitro.
Protein liquid crystal from a biological system
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A. C. Neville and B. M. Luke
Fig. 8. Electron micrograph of oblique section of 5. tenuidentata oothecal protein
spherulite. The parabolic patterns have lamellar periodicity and are formed by the
electron-light component. The heavily stained matrix is the liquid crystalline continuous phase, x 15000.
Fig. 9. As for Fig. 8 but heated to 55 °C before fixing. The matrix has formed a gel and
both lamellae and patterning have disappeared, x 15000.
Protein liquid crystal from a biological system
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Fig. io. Electron micrograph of M. monacha colleterial gland. The end apparatus of
a gland cell dominates the field, and is lined with microvilli (/c, luminal cells; hi, lumen
of gland tubule). The gland cell is packed with rough endoplasniic reticulum, and the
cell is not actively secreting, x 6500.
Fig. 11. As for Fig. 10 but for an actively secreting gland cell. Note that the parabolic
patterning which characterizes a helicoid is not seen either in the end apparatus of
the gland or in the discharging protein. It first appears by self-assembly several microns
from the cells, at p. x 11 500.
Protein liquid crystal from a biological system
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A.C. Neville and B. M. Luke
Fig. 12. Electron micrograph of colleterial gland cell cytoplasm of M. monacha. Much
ribosome-studded endoplasmic reticulum is present, correlated with the synthesis of
large volumes of oothecal protein for export. Vesicles, v, are thought to contain the
protein en route to the end apparatus, ea. x 19000.
Fig. 13. Electron micrograph of inside edge of wall of colleterial gland from M. monacha.
Note the absence of a deposition zone by contrast with insect cuticle deposition,
(e, epicuticle lining of luminal cell; p, parabolic patterning in liquid crystalline protein
within lumen. Arrows indicate convoluted cell membrane separating luminal cell,
Ic (containing microtubules), from gland cell, gc (containing rough endoplasmic reticulum).) x 27000.
Fig. 14. Electron micrograph of the edges of oothecal protein spherulites from
M. monacha. x 8500.
Protein liquid crystal from a biological system
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13