|Checklist of the Collembola: Some notes on the Ultrastructure of the Cuticula|
This page is under construction.
The major part of the information presented here is common knowledge compiled from the literature and from personal communications with other researchers, and it is not based on research results from our lab. The added value comes from the synergic application of concepts compiled from different disciplines.
All different concepts of the disciplines involved are shortly introduced. Specific Collembola related issues are mentioned when applicable.
The epidermis is a simple, unstratified epithelial tissue.
It consists of a single layer of polyhedral cells.
Depending on the life cycle phase of the animal
the cells are squamous, cuboidal or columnar,
with or without a microvillar apical surface.
The extracellular cuticula is differentiated from outside to inside into the epicuticula, the procuticula or exocuticula, and the subcuticula or endocuticula.
The outer surface of the cuticula is seldom smooth or bare;
it presents a great variety of microscopic roughenings in the form of points,
pits, ridges, and sculptured
designs, and it is covered with larger outgrowths that take the shape of
spicules, spines, hairs, and scales.
All the external processes of the body wall, however, may
be classed in two groups according to whether the epidermal cells take a
direct part in their production or do not;
that is, they are either cellular or noncellular
outgrowths. Of the cellular processes, some are unicellular,
others are multicellular (Snodgrass, 1935).
The epidermis is a monolayer of matrixcells
responsible for producing at least part of the basement membrane
as well as all of the layers of cuticula.
It is seen in its full development only when the new cuticula is being produced
(Wigglesworth, 1965:25). When it is fully active the apical part of the
epidermal cell is striated, with microvillar extensions into the vertical
pore canals of the cuticula.
In the growing stages of
insects the epidermal cells are usually cubical or columnar,
with the nuclei near their bases;
but in adult insects, after the activity of cuticula formation is over,
matrix cells become more or less degenerate and
appear in most places as a thin protoplasmic layer beneath the cuticula,
in which cell boundaries are indistinct and
the cell areas are marked only by the nuclei (Snodgrass, 1935).
The histological appearance of the cuticula varies somewhat in different insects and in different parts of the integument of the same insect. The endocuticula has a faint horizontally lamellate structure, in which usually there are visible fine vertical striations. The striations appear to be canals left by protoplasmic filaments that, during the formative stage of the cuticula, extend outward from the epidermal cells. The cuticular material is probably laid down in layers between these filaments, which are later retracted. N. Holmgren (1902) has suggested that the protoplasmic strands of the epidermis represent primitive cilia that once may have covered the bodies of the arthropod ancestors (Snodgrass, 1935).
The basement membrane (lamina basalis) serves as a backing for the epidermal cells and effectively separates the hemocoel (the main body cavity) from the integument.
The cuticula is formed by an apical epidermal cell secretion of water, proteins, chitin and lipids.
The cuticula differentiates in a hard exocuticula, an optional mesocuticula and a soft endocuticula. Differentiation of the exocuticula involves a chemical process (called sclerotisation) that occurs shortly after each moult. During sclerotisation, individual protein molecules are linked together by quinone compounds. These reactions "solidify" the protein matrix, creating rigid "plates" of exoskeleton known as sclerites. After moulting a hormone bursicon stimulates the epidermal cells to secrete phenolic compounds which permeate the cuticule, undergo oxidation and due to action of phenolases cross-link cuticular proteins. Quinone cross-linkages do not form in parts of the exoskeleton where resilin (an elastic protein) is present in high concentrations. These areas are membranes -- they remain soft and flexible because they never develop a well-differentiated exocuticula.
The epidermis is primarily a secretory tissue formed by a single layer of epithelial cells. The basement membrane is a supportive extracellular matrix of amorphous mucopolysaccharides, the basal lamina, enforced with an embedded reticular layer of collagen fibers. The cuticula lies immediately above the epidermis. It contains microfibers of chitin surrounded by a matrix of protein that varies in composition from species to species and even from place to place within the body of a single specimen.
The succession of ecdyses divides the life span of the animal into a series of stages, while the animal itself appears as a series of instars. The number of moults varies with different species and is frequently different with individuals of the same species reared under the same conditions. It is influenced somewhat by temperature, humidity, and the amount of feeding. Yet, notwithstanding all irregularities, the number of moults is surprisingly constant for each species and may be characteristic of families and even orders (Snodgrass, 1935).
The beginning of an instar is not marked by the discarding of the old cuticula, though in "life-history" studies the length of a developmental stage is usually measured from the time the exuviae are cast. Physiologically, however, it should be reckoned from the time the old cuticula is loosened from the epidermis, which more approximately marks the beginning of the short period of development that is to give the increased size and the characteristics of the following instar. The loosened cuticula may not be shed for several days. When the cuticula begins to separate from the epidermis preparatory to ecdysis, the insect usually ceases to feed and becomes more or less quiescent. Each active stage in the insect's life is thus followed by a sluggish premoulting period (after Snodgrass, 1935).
The moulting process is triggered by hormones released when a Collembola's growth reaches the physical limits of its exoskeleton. Each moult represents the end of one growth stage (instar) and the beginning of another. Moulting does not stop when the Collembola becomes an adult: about 20 moults in Pseudosinella from caves (Barra, 1991:191), about 67 moults in Hypogastrura viatica kept in experimental conditions at 20 degrees Celcius (Mertens & al., 1983).
A new, larger exoskeleton is constructed inside the old one. The moulting process begins when epidermal cells respond to hormonal changes by increasing their rate of protein synthesis. This quickly leads to apolysis -- physical separation of the epidermis from the old endocuticle. In Podura aquatica the old cuticle is lifted away from the epidermal cells by a foam-like secretion (Wiggelesworth, 1965:41). The epidermal cells initially secrete the inactive precursors of chitinase and protease, followed by cuticulin, a highly cross-linked protein that is deposited in the form of a set of layers. Cuticulin itself, formed by phenolic polymerisation of arthropodin, a mixture of soluble proteins, is resistive to enzymatic hydrolisation. The cuticulin matrix insulates and protects the epidermis from the moulting fluid's digestive action that becomes active only when the new cuticulinar multilayer is complete. The hydrolytic enzymes are too large to get through the mazes of the cuticulinar matrix. They hydrolise the proteins of the old endocuticula and the products of this digestion, amino acids and glucosamine units, that are small enough to get through the mazes of the cuticulinar matrix are recycled by the epidermis to form the new cuticula.
The separation of the old cuticula
from the epidermis is accomplished by a moulting liquid formed
by the epidermal cells, and/or by special exuvial glands of the epidermis,
that dissolves the inner layers
of the endocuticula and thus frees the rest of the cuticula from
the epidermis (after Snodgrass, 1935).
In Collembola, which moult during the adult stage,
the exuvial glands are said to persist throughout life.
Barra, 1973:(Figure 17.C) confirmed the existance of the exuvial glands in Collembola.
The post-apolysis exuvial fluid of Collembola is characterised by the presence of proteineous polysaccharidic moulting granules (Barra, 1970:3243) and specialised haemocytes - also called ecdysohaemocytes or exuviocytes (Barra, 1991:191). The release of moulting granules into the exuvial fluid by tormogen cells is followed by the protrusion of the the exuvial lumen by thrichogen cells. Two types of ecdysohaemocytes can be distinguished: the typical granulocyte and a granulocyte with bilobed lysosomes. Apparently, the inactive moulting granules are activated by granules released by the granulocytes. The moulting granules and ecdysohaemocytes may play a role in the cuticular digestion (Barra, 1991:191).
The intermoult period can be divided - based onto
the moulting granule lifecycle - into a
pro-exuvial phase followed by a post-exuvial phase of equal duration
- pro-exuvial phase: after apolysis, the moulting granules released from the tormogenic vacuole into the exuvial fluid migrate towards the basis of the old endocuticula; the epicuticula is formed by the epidermal cells
- end of pro-exuvial phase: the old endocuticula and exocuticula is digested and the number and size of the moulting granules is reduced; the new exocuticula is formed by the epidermal cells; the tormogen cells form the setal base membranes
- post-exuvial phase: the microvilli of the tormogen cells retract, creating extracellular vacuoles at the setal bases
- end of post-exuvial phase: the moulting granules produced by the tormogen cells migrate into the extracellular vacuole; the new endocuticula is formed by the epidermal cells
|Day||Apolysis||Pre-exuvial period||Exuvation||Post-exuvial period|
|Moulting granules in exuvial space||Deposition of epicuticula||Deposition of exocuticula||Ecdysis||Deposition of endocuticula||Secretion of moulting granules|
After formation of the cuticulin layer, the moulting fluid becomes activated and chemically "digests" the endocuticula of the old exoskeleton. Break-down products (amino acids and saccharides) pass through the cuticulin layer where they are recycled by the epidermal cells and secreted under the cuticulin layer as new procuticula (soft and wrinkled).
Generally, it is accepted that the
transcuticular pore canals
transport the liquid lipids and proteins to the new epicuticula
where the wax
layer is formed.
But this can be questioned...
Apparently, these pore canals are the remnants of the epidermis cell microvilli (see Barra, 1973:(Figure 23): an oblique transversal section through the procuticula of Tomocerus minor clearly shows the actin filaments of the microvillar cytoskeleton in the so-called pore canals). After ecdysis, the epidermal cells retract their microvilli from the newly deposited procuticula. This retraction creates a vacuum in the left behind 'empty' pores of the procuticula. Due to the suction, as a result of the vacuum, the pores are filled with the liquid wax phase of the epicuticula. This proposal assumes that there is an alternative mechanism present that deposits the wax layer on top of the epicuticula (specialised glands?).
Even without a wax layer the Collembolan cuticula is relatively impermeable (Ghiradella & Radigan, 1974:305). The wax layer is not permeable to water, but it is to oxygen or atmospheric gasses in general. The very thin (optional) polyphenolic layer between the cuticulin layer and the wax layer is not permeable to gasses.
The epicuticula's main function is to reduce water loss.
When young animals of Hypogastrura viatica, cultured submerged in water,
are transfered from water to air, the cuticula becomes irreversibally
hydrophobe and they cannot be resubmerged.
From the age of three weeks after hatching, however, the animals do not
survive the transfer to air (Mertens & al., 1983:576).
This suggest that the wax layer is formed by the first instars
only when it is required: when cultured submerged in water,
water loss is not a problem and the wax layer has no function.
The waxed epicuticula of Pogonognathellus flavescens is not wetted by water at less than 2.5 atmosphere pressure (Ghiradella & Radigan, 1974:305).
When the new exoskeleton is ready, muscular contractions and intake
of air cause the body to swell until the old exoskeleton
splits open along lines of weakness, ecdysial sutures.
The animal sheds its old exoskeleton (ecdysis)
and continues to fully expand the new one.
Over the next few hours, sclerites will harden and darken as
quinone cross-linkages form within the exocuticle.
This process, called sclerotisation or tanning,
gives the exoskeleton its final texture and appearance.
The cement (shellac) layer is secreted by dermal glands after moulting.
An animal that is actively constructing new exoskeleton is said to be in a pharate condition. During the days or weeks of this process there may be very little evidence of change. Ecdysis, however, occurs quickly (in minutes to hours). A newly moulted springtail is soft and largely unpigmented (white or ivory). It is said to be in a teneral condition until the process of tanning is completed (usually a day or two).
To be completed.
1. reduction of microvilli precedes secretion
2. cells secrete monomer cuticulin by reverse pinocytosis (merocrine exocytosis) or by apocrine secretion of the microvillar apices (see 1)?
3. due to polyphenolic tanning (=formation of hydrogen cross links between protein chains) the monomers self-assemble into monolayers or spherical micellae
4. micellae self-assemble into particle films onto the microvillar apices of the epidermal cells into a pattern that is superimposed on the spatial microvillar pattern: the origin of the hexagonal epicuticular texture.
While the new exocuticula and endocuticula is formed, the microvilli themselves gradually extend, and the exocuticular and endocuticular micellae are deposited into the intermicrovillar space: from the spatial microvillar pattern also the so-called transcuticular pore canals originate.
1. tightly packed, stub-shaped microvilli
2. microvillar height, array, architecture
3. inter-microvillar spacing
4. glycocalyx or 'slime layer' = membrane carbohydrates that bind excreted products of cell metabolism
To be completed.
The surfaces of cells are covered with
glycoproteins, glycolipids, and proteoglycans,
forming a dense layer called the glycocalyx.
This covering provides a
protective barrier for the cells and serves as a
supporting matrix for secretions. In addition, the individual
glycoconjugates bind to growth factors, enzymes, and adhesive proteins,
and thereby participate in a
wide variety of biological phenomena related to cell differentiation,
proliferation, tissue formation and morphogenesis. To a large extent
these interactions are
determined by the structure and binding properties of the polysaccharide
chains (glycans) that distinguish glycoconjugates from other types of
The study of the structure, function, and metabolism of glycoconjugates defines the field of glycobiology. Interest in this field is rapidly expanding, since it is clear that to understand macromolecular function requires an appreciation of glycosylation.
These molecules are common to all multicellular organisms, and are found on the surface of cells, in secretory granules and in the extracellular matrix. Most cells produce several types of proteoglycans, usually containing chains belonging to the heparan sulfate or chondroitin sulfate families of glycosaminoglycans. The glycosaminoglycans bear a strong negative charge due to hexuronic acid epimers and sulfate groups. The arrangement of sulfate groups and the epimers promote specific protein-carbohydrate interactions, which affect fundamental properties of cells, such as adhesion and endocytosis.
Apinhasnit & al. (1988) showed that the conventional method of tissue preparation for TEM did not preserve the glycocalyx in its entity. They concluded that the glycocalyx consists of two parts, an inner continuous layer which is tightly bound to the apical plasma membrane and is always preserved, and an outer filamentous layer which is not always preserved by the conventional method. The glycocalyx is mainly a negatively charged glycoprotein matrix with a substantial amount of many types of sugar residues: such as a-D-mannose, a-D-glucose, N-acetyl-glucosamine, N-acetyl-neuraminic (sialic) acid and b-D-galactose. This negatively charged glycocalyx provides a receptor site for ...
The cell coat is a layer of carbohydrates on the surface of the cell membrane. It is made up of the oligosaccharide side-chains of the glycolipid and glycoprotein components of the membrane and may include oligosaccharides secreted by the cell.
Paustian (2000) attributes the following functions to the glycocalyx:
attachment to environment;
reservoir for certain nutrients: the glycocalyx will bind certain
ions and molecules that can then be made available to the cell;
depot for excreted products: excretions will accumulate in the
glycocalyx, that binds them up.
The glycocalyx serves two important purposes. First, its oligosaccharide and polysaccharide constituents absorb water to form a slimy surface. This surface protects the membrane from mechanical and chemical damage and prevents the cell from sticking nonspecifically to substrates. Second, the glycocalyx functions as the cell's uniform. Particle-cell adhesion occurs by recognition of specific carbohydrate moieties in the glycocalyx. Proteins that recognize carbohydrate sequences are called lectins.
Brown & al (1981) found three predominant glycoproteins in the rat thymocyte plasma membrane. Two of these have carbohydrate compositions that are characteristic of structures N-glycosidically linked to protein. The other glycoprotein is very different, having about 20 O-glycosidically linked carbohydrate units per 100 amino acids.
The epidermal microvilli have
projections of glycoprotein molecules which are termed the glycocalyx.
The sugars from heavily glycosylated membrane proteins
and from glycolipids in the plasma membrane form the glycocalyx,
a thick carbohydrate
layer that covers the cell surface.
This glycocalyx has enzymatic properties, like saccharidase,
alkaline phosphatase and aminopeptidase.
These glycoprotein enzymes have a hydrophobic end imbedded
in the lipid layer of the cell membrane
and a hydrophilic end projecting into the exuvial lumen.
This hydrophilic end contains the particle binding site.
The glycocalyx enables absorption of water,
minerals, amino acids and simple sugars from the exuvial space.
The glycocalyx enables the cell to adhere particles to its surface.
Glycocalyx formation: The cell coat is a secretion product incorporated into the cell surface that undergoes continuous renewal. Glycocalyx glycoproteins are regularly replaced via biosynthesis in the ribosomes of the rough endoplasmic reticulum, followed by final assembly with the oligosaccharide moiety in the Golgi apparatus, that subsequently packs them into membrane bound secretion granules, that get integrated in the plasma membrane.
Biological membranes are asymmetric with respect to lipid composition and transmembrane protein orientation. Lipid synthesis and ribosomal transmembrane protein synthesis takes place on the cytosolic surface of the endoplasmic reticulum. Glycosylation of lipids and transmembrane proteins occurs in the lumen of the endoplasmic reticulum and in the lumen of the Golgi complex. Mature lipids and proteins are delivered to the plasma membrane by vesicles. Through the events of vesicle extrusion and fusion, the interior surface of the endoplasmic reticulum and Golgi complex membranes are topologically equivalent to the exterior surface of the cell.
The high density of charged hydrophilic oligosacharide side-chains of glycoproteins of the glycocalyx region retains a layer of immobile water. The plasma membrane exhibits a glycocalyx, consisting of carbohydrates anchored to the membrane bilayer. The morphological features of this glycocalyx differ in function of its constituents. A dense, continuous layer of glycolipids extends 10-20 nm from the phospholipid-water interface. A more fibrous glycocalyx is made of glycoproteins and/or proteoglycans typically measuring 5-8 nm thick and 100-200 nm long consisting of 10,000 atoms or more.
Plasma membranes are mechanically supported by the cell cortex on their cytosolic surface, and are protected by the glycocalyx on their exterior surface. The sole function of glycophorin, an abundant membrane protein in erythrocytes (red blood cells), is to participate in the glycocalyx and cortex structures.
Carbohydrate makes up 2 to 10% of the plasma membrane by weight. It is exclusively on the outer surface of the cell; the fuzz of carbohydrate is called the glycocalyx. Membrane carbohydrate comes in three basic forms.
Glycolipids are lipid molecules with covalently attached sugar groups: these are found only in the outer leaflet. More than 30 different kinds of glycolipids have been identified. The most complex glycolipids are gangliosides, which contain one or more sialic acid residues. In turn, more than 30 different gangliosides are known: their functions are unknown, but one acts as a cell surface receptor.
Glycoproteins are proteins with covalently attached short chains of sugar groups, called oligosaccharides. Some of these are integral to the membrane, others are adsorbed.
A final form of membrane carbohydrate is the proteoglycans, which are adsorbed to the membrane. Proteoglycans differ from glycoproteins in being dominated by carbohydrate (they are 90-95% carbohydrate by weight), and are therefore much larger, with long, unbranched side chains, called polysaccharides. Proteoglycans were formerly called mucoproteins. The carbohydrate part of the molecule is called a glycosaminoglycan (GAG), it was formerly called a mucopolysaccharide. The GAG and the protein portions of a proteoglycan are covalently linked.
Transmembrane proteins, also called integral membrane proteins. Ion channels are integral membrane proteins that can be present in as few as 50 copies in an entire cell. The combination of complexity and rarity means that transmembrane proteins are difficult to study.
Glycophorin was the first membrane protein to be sequenced. It is a glycoprotein, with 131 amino acid residues in the protein portion and 16 oligosaccharide side chains carrying almost 100 sugar residues. Each red blood cell has 600,000 glycophorin molecules.
Although most of the carbohydrate is attached to intrinsic plasma membrane molecules, the glycocalyx usually also contains both glycoproteins and proteoglycans that have been secreted into the extracellular space and then adsorbed onto the cell surface. (Alberts & al., Molecular Biology of the Cell, 3rd ed., p.502)
As the oligosaccharides and polysaccharides in the glycocalyx adsorb water, they give the cell a slimy surface. This helps as lubrication during apolysis.
Knudson (1998) found that tumor cells have the capacity, when in the presence of binding proteoglycans, to assemble components into a pericellular matrix shell or coat. Analogous to a bacterial glycocalyx, this pericellular coat may serve to cocoon the malignant cells.
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Self-assembly of amphiphilic molecules.
- self-assembled monolayer of amphiphilic molecules.
- self-assembled bilayer of amphiphilic molecules.
- micella: self-assembled structure of amphiphilic molecules in a polar liquid.
- inverted micella: self-assembled structure of amphiphilic molecules in a non-polar liquid.
Lipid-protein complexes, lipoproteins have a micellar structure, with a spherical core of triacylglycerols and cholesterol esters having a coating of phospholipids, cholesterols and apolipoproteins.
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