Optical microscopy

Optical microscopy study

Técia Ulisses de Carvalho

Biophysics Program, Carlos Chagas Filho Biophysics Institute, Federal University of Rio de Janeiro

Email: tecia@biof.ufrj.br

The first morphological descriptions of Trypanosoma cruzi by Carlos Chagas were made by observing the fixed parasite, stained with Giemsa, a method that is still used to this day. Optical microscopy allows us to identify, in the parasite: (i) the general shape of its cell, (ii) its nucleus, (iii) kinetoplsat (condensed mitochondrial DNA, always located near the flagellum in trypanosomatides). Its name comes from the early belief that this structure (kineto = motion; plast = organelle) was responsible for flagellum motion and (iv) flagellum.

According to: (i) the general form of the evolutionary form; (ii) the relative position between the flagellum and the nucleus; (iii)the location of the flagellar pocket (from which the flagellum emerges); and (iv) location of the free flagellum, it is possible to identify the different evolutionary forms of trypanosomatides.

In the case of Trypanosoma cruzi, observing the parasite through optical microscopy allows us to identify three well defined forms:Tripomastigote: elongated form (which can present as slender or stumpy), with a round kinetoplast in the area posterior to the nucleus, emerging from the flagellar pocket (not visiblle through optical microscopy), which is located laterally, in the posterior region of the parasite. The flagellum emerges and adheres longitudinally to the body of the parasite, becoming free in the anterior region. This form is highly infective and can be found in the vector insect (posterior portion of the gut, in the rectum), in the blood and in the intercellular space of vertebrate hosts, in cultures of infected cells, and in axenic cuultures (in vitrometacyclogenesis).

Amastigote: rounded shape, with bar or rod-shaped kinetoplast in the region anterior to the nucleus, short flagellum (not visible to optical microscopy) emerging from the flagellar pocket. This form can be found inside cells in infected hosts, as well as in axenic cultures.

Epimastigote: elongated form, with bar- or rod-shaped kinetoplast located in a region anterior to the nucleus. The flagellum emerges from the flagellar pocket with a lateral opening and runs along the body of the parasite, adhered to it; it becomes detached in the anterior region. Can be found in the digestive tube of the vector insect and in axenic cultures.

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Electron microscopy

Study using scanning electron microscopy

Wanderley de Souza and Juliana Vidal 

Carlos Chagas Filho Biophysics Institute, Federal University of Rio de Janeiro 

Email: wsouza@biof.ufrj.brImages obtained through scanning electron microscopy provide us with information about the surface of the specimen being studied, generating images with morphological information that is important for taxonomic and ultrastructural studies about many organisms, and shedding light on cell-cell interaction. As previously mentioned, Trypanosoma cruzi has three development stages, each of which has a characteristic cell shape (Figure 1) widely studied by optical and scanning electron microscopy.

Study using transmission electron microscopy

Wanderley de Souza and Juliana Vidal

Carlos Chagas Filho Biophysics Institute, Federal University of Rio de Janeiro

Email: wsouza@biof.ufrj.brTransmission electron microscopy has been widely used in all areas of biological and biomedical sciences, for its ability to display the thinnest of cellular structures, therefore making it possible to study cellular organization. Trypanosoma cruzi has organelles that are typical of eukaryotic organisms, such as mitochondria, peroxysomes and lysosomes, as well as endoplasmic reticulum, while others are peculiar to them. Figure 1 is a general diagram of the epimastigote form of T. cruziwhich will serve as foundation for a summarized description of the main components.

Cellular surface

When analyzed through a transmission electron microscope, the surface of all forms of T. cruzi has a discrete glycocalyx approximately 7 mm thick. This glycocalyx actually consists of carbohydrates that project outwards from the cell, and are associated to peripheral or integral proteins, forming glycoproteins, and to lipids, forming glycolipids (Figure 2). Surface studies using the “fracture-flip” technique show that the epimastigote form is quite smooth, except for the most specialized region of the cytostome (Figure 3), when compared with the surface of amastigote and trypomastigote forms (Figure 4), which present, when observed with the same technique, a rougher surface. The morphological differences between the surface of epimastigote and trypomastigote forms observed through the fracture-flip technique possibly reflect differences in mucin size, as GPI-anchored mucins in trypomastigotes are larger than those found in epimastigotes.

Plasma membrane

The plasma membrane that involves the cells plays an important role as it separates the extracellular from the intracellular environment, working in a highly selective and specific way as it functionally interconnects these two compartments. As in all cells, the plasma membrane of T. cruzi has a double lipidic layer to which proteins of different natures are associated, in different degrees.The presence of proteins in the plasma membrane of T. cruzi has been analyzed with structural and biochemical techniques. With the use of structural techniques such as cryofracture, it was possible to demonstrate that there are various dominions in the plasma membrane that surrounds T. cruzi and other trypanosomatides, and that there are significant differences between the different evolutionary forms. The images obtained with the conventional cryofracture technique show integral membrane proteins that appear in transmission electron microscopy in the shape of small particles that were named intramembranous particles. Thanks to this technique it was possible to demonstrate that the membrane that encases epimastigote forms is much richer with integral proteins than the one that enclosed the amastigote form, and this one is larger than the one that encases the trypomastigote form. On the other hand, special membrane areas, presenting different characteristics from the membrane that encases the cellular body, were also found (Figure 5). The membrane that lines the flagellum is very poor in intramembranous particles, except for the region at the base of the flagellum, where there is an agglomeration of particles originating a structure called ciliary necklace, and in the areas of adhesion between the flagellum and the cellular body, where particles have a more linear arrangement, configuring an area of junction of the flagellum and the cellular body. This junction involves the organization of intramembranous particles found in the flagellar membrane in the epimastigote forms (Figure 5) and both in the flagellar membrane and in the membrane that encases the cellular body in the case of trypomastigote cells, suggesting a much more efficient adhesion in this latter form. So far, junctional proteins have not yet been characterized. However, a series of high molecular weight proteins that are highly antigenic have been identified using immonocytochemical methods, in the flagellar adhesion zone (FAZ), where the flagellum adheres to the cellular body. Another well characterized protein that seems to be involved in the adhesion between flagellum and the cell of the epimastigote form is a glycoprotein (72 kDa), known as Gp 72. The knock-out of the gene that codifies this protein led to the appearance of a form in which the flagellum does not remain linked to the cellular body. This situation was then reversed by introducing the gene via transfection.

The epimastigote and amastigote forms of T. cruzi capture nutrients through endocytosis by means of two portals: the flagellar pocket and the cytostome-cytopharynx complex. These are both invaginations of the membrane, representing a specialized membrane domain. The cytostome is an opening on the anterior region of the surface of epimastigotes, followed by an invagination of the membrane called cytopharynx. It is accompanied by seven microtubules, of which four originate near the flagellar pocket and pass beneath the pre-oral rim before they reach the cytopharynx.The flagellar pocket of T. cruzi has four specialized microtubules near the basal body (a cylinder-shaped structure consisting of nine triplets of microtubules, present in the cell cytoplasm, whose function is to direct tubulin polymerization to form the flagellum), just like in T. brucei (Figure 6).

Since the advent of transmission electron microscopy there have been breakthroughs in microscopy technologies and in the preparation of samples for observation using this method. One of them is a technique called electron tomography. In general terms, this technique is similar to the computerized tomography used in the medical field, and generates three-dimension images of the cell being studied.Using this kind of microscopy, 3D analysis of the cytostome-cytopharynx complex of T. cruzi showed, for the first time, that in epimastigote forms this complex is surrounded by a set of seven microtubules, arranged in a “gutter” shape around the cytopharynx. In addition, it was possible to observe aligned vesicles near the free region of the cytopharynx, an indication of fusion or budding of vesicles in this invagination (Figure 7).

Cytoskeleton

The four main components of the cytoskeleton of cells, (i) microtubules, (ii) thick filaments, (iii) intermediary filaments

and (iv) microfilaments, play an important role in the maintenance of the shape of eukaryotic cells, cell movements, intracellular movements, control of mobility of membrane components etc. Studies of the cytoskeleton of T. cruzi are still incipient. Practically nothing is known about thick and intermediary filaments. Microfilaments have not been identified yet, in spite of a great number of structural studies already done. However, actin, the main component of microfilaments, has already been identified in T. cruzi, using immunofluorescence microscopy as well as biochemical studies. On the other hand, microtubules have been studied in more detail and represent the main component of trypanosomatide cytoskeleton.

T. cruzi has different cytoskeleton and cytoskeleton-related structures that are crucial for the biology of the protozoan, such as the subpeculiar microtubules layer (SPMT), which are stable microtubules responsible for the cellular format that is typical of trypanosomatides, as they form a sort of helicoidal framework located right beneath the plasma membrane (Figure 6). Subpeculiar microtubules are resistant to low temperatures and do not suffer significant depolymerization when treated with powerful mammal microtubule destabilizers, although they are not insensitive to these drugs. It is believed that the plasma membrane and subpeculiar microtubules form a structural and functional unit, the periplast, responsible for maintaining the characteristic shape of the cell and for all the activities regarding the interface between the protozoan and the external medium.In the plasma membrane, subpeculiar microtubules are absent only in the region where the flagellum adheres to the cellular body, in the flagellar pocket, and in the cytostome. For this reason, the latter two are the only sites where the nutrients necessary for cellular metabolism are captured through the endocytosis process.

Flagellum

An important structure in trypanosomides is the single flagellum, which emerges from the body through an invagination of the plasma membrane called flagellar pocket. The extremity of the cell from which the flagellum becomes free from the body is the anterior one. In the amastigote form, the flagellum is very short, very often remaining within the flagellar pocket. For this reason, sometimes it is not seen through optical microscopy, which led to the creation of the term amastigote (without flagellum) to incorrectly describe the multiplicating intracellular form of T. cruzi and of Leishmania.

The axonem of the flagellum is formed by the standard arrangement of nine pairs of peripheral microtubules and one central pair. The flagellum is closely associated to a basal body, a structure firmly linked to the cell cytoplasm and typically presenting nine peripheral triplets of microtubules (Figure 7). An interesting fact is that the basal body of trypanosomides has small filaments joining it to the kinetoplast (a region of the mitochondrion where mitochondrial DNA is confined) and maintains the kinetoplast perpendicular to the axis of the flagellum.

The flagellum of trypanosomides is kept adhered to the cellular body of the protozoan by the flagellar attachment zone (FAZ). Some studies have used experiments that make it possible to delete genes and therefore prevent the translation of certain proteins of interest. When FAZ proteins were deleted, the result was parasites with defects in flagellum morphogenesis and with deficient endocytosis. An interesting fact is that the basal body of trypanosomides has small filaments joining it to the kinetoplast (a region of the mitochondrion where mitochondrial DNA is confined) and maintains the kinetoplast perpendicular to the axis of the flagellum. The interphase cell also has an accessory basal body, connected to the first by filamentose structures that have not been characterized yet.On the axonem side, the flagellum of the epimastigote and trypomastigote forms has a complex structure called paraxial body or paraflagellar rod (PFR) (Figure 7). The PFR is a structure present exclusively in kinetoplastids, except in those that have an endosymbiont bacterium. The best described function of the PFR is its participation in the mobility of kinetoplastids. This structure consists of a complex arrangement of filaments of different thicknesses (25 and 70 nm) that are connected by special bridges to pairs 4 and 7 of microtubules of the axonem. Biochemical studies indicate the presence of two major proteins (69 and 80 kDa), and various other minority proteins, as part of the paraxial structure. Mutants ofT. brucei and C. fasciculata that do not express a gene that codifies one of the major proteins of the paraxial structure show a significant reduction in motility. The PFR has immunological relevance: a study recently showed the mechanism through which one of the paraflagellar proteins is exposed by means of class I MHC, making it possible for T lymphocytes to recognize infected cells and control the infection by T. cruzi.

In the case of epimastigote and trypomastigote forms, part of the flagellum is adhered to the cellular body, establishing a kind of junction as described above. The fact that this firm association exists results in a certain movement of the body when the flagellum lashes, giving the impression of an undulating membrane. However, tryponasomatides do not have a structure that can be identified as an undulating membrane, such as what happens in trichomonads, for instance.

It should be noted that as it protrudes from the flagellar pocket, the flagellum membrane establishes a very close contact with the membrane that lines the transition area between the flagellar body and the flagellar pocket. This contact is important as it establishes a certain communication barrier between the medium and the flagellar pocket.

The motion of the flagellum happens thanks to a sliding movement between the axonem microtubules, with the participation of a special protein with ATPasic action, called kinesin. In general terms, the beginning of a wave that propagates along the flagellum occurs in one of the extremities. If the wave begins at the tip of the flagellum and propagates towards its base, the cell moves forward, and we can say the flagellum is drawing the cell. When the wave begins at the base of the flagellum and propagates to its tip, the movement happens in the opposite sense, and we can say the flagellum is pushing the cell. In general, flagellated eukaryotic cells only execute one of these movements. Trypanosomatides, however, have both movements, which makes them extremely efficient in directing their motion.An important role played by the flagellum of T. cruzi is making it move. This movement is crucial at the end of the intracellular cycle, when trypomastigote forms are formed and move intensely in the cytoplasm of the host cell. This motion appears to be responsible for breaking the cell, already altered, and for releasing trypomastigotes into the intercellular space. These trypomastigotes must migrate to interact with new cells or to reach the bloodstream. As we will see later, in the moment the trypomastigote comes into contact with a new cell of the vertebrate host and during the interaction process between the epimastigote forms and the wall of the digestive tube of the invertebrate host, the flagellum seems to play an important role.

Kinetoplast

Trypanosomatides have a single mitochondrion that is ramified and extends all along the cellular body. The class to which these protozoa belong is named Kinetoplastea after one of the typical characteristics of trypanosomatides, the presence of the kinetoplast. The kinetoplast is a specific region of the mitochondrion where the mitochondrial DNA (kDNA) is concentrated. This DNA is formed by a wide network of interconnected circular molecules. This arrangement represents approximately 30% of the total DNA of trypanosomatides. The kinetoplast is basophile and can easily be observed through optical microscopy, in preparations stained with Giemsa.This special region of the mitochondrion is located near the basal body, perpendicularly to the axis of the flagellum, and is physically connected to the basal body by filaments. The format of the kinetoplast and its position regarding the nucleus are criteria usd to classify the different development forms of T. cruzi. These forms are: epimastigote, amastigote, and trypomastigote (Figure 8). 

Glycosomes

Following the first studies of the ultrastructure of trypanosomides, scientists observed the presence of an organelle encased in a membrane 6 nm thick and with a relatively dense matrix, which fit the general concept of microbodies. In T. cruzi, and in most trypanosomides, it is spherical in shape and has a diameter of about 0.3 mm. The microbodies found in various eukaryotic cells have been identified as organelles involved in the unfolding of peroxyde, through the action of catalase, and in the oxidation of amino acids, receiving the designation of peroxisomes. Cytochemical studies did not show the presence of catalase in the microbodies found in T. cruzi. However, other inferior trypanosomides presented a positive reaction.

As mentioned before, the blood form of T. brucei practically does not have a mitochondrion, but it does have a large amount of microbodies. This fact allowed Opperdoes and collaborators to isolate this organelle and to proceed with its biochemical characterization, showing that practicallly all enzymes of the glycolytic pathway of the protozoan were located in it, reaason for which he named it glycosome. Shortly after, these observations were confirmed in various trypanosomatides, including T. cruzi. Cytochemical studies have shown that glycosomes had a great quantity of basic proteins, which was later explained with the observation that the enzymes of the glycolytic pathway located in the organelle have an alkaline isolectric point, making it different from those found in the cytoplasmatic matrix of eukaryotic cells. Glycosomal proteins are synthetized in cytoplasmatic ribosomes and are then transferred, with the participation of a signal peptide, into the glycosomes. Based on the available data, we can consider glycosomes a specialized type of peroxisome existing in members of order Kinetoplastida. In addition to the enzymes of the glycolytic pathway, this organelle has other enzymes that participate in catabolic and anabolic processes. Many of these enzymes are crucial for the proliferation, viability and virulence of the parasites. For this reason, many inhibitors of these enzymes have been developed and have proven to be effective as inhibitors of T. cruzi growth. Several studies have shown that the integrity of the glycosomes and the proper compartmentalizatioun of the enzymes that make up the matrix of this organelle are crucial for parasite viability.

Endoplasmic reticulum

Profiles of smooth and rough endoplasmic reticulum can be found all over the body of all evolutionary forms of T. cruzi. There is often a higher concentration of reticulum in the peripheral area, near the subpeculiar microtubules (Figure 9). Ribosomes are distributed all over the cytoplasm, often organized in the form of polysomes.The Golgi apparatus is always present, located between the nucleus and the kinetoplast. In general, it consists of a system of 3 to 10 cysterns and vesicles in the trans portion. In forms that have a cytostome-cytopharynx (Figure 9B), such as epimastigotes and amastigotes, it is located near this structure. Cytochemical studies have shown that the Golgi apparatus of T. cruzi participates in the glycosylation process of glycoconjugates.

Reservosomes

The epimastigote and amastigote forms of T. cruzi can ingest macromolecules from the medium, via endocytosis. This activity is either non-existent or very low in the trypomastigote form. The use of tracers that can be observed through transmission electron microscopy (either directly, as is the case of proteins associated to colloidal gold particles, or through cytochemical detection, as is the case of peroxidase) made it possible to establish the endocytosis pathways and the destination of the endocytosed material. When epimastigotes are incubated in the presence of macro molecules, these are interiorized by two regions only: the flagellar pocket and the cytostome-cytopharynx. In the cases in which endocytosis is mediated by receptors, such as what happens with the interiorization of LDL and transferrin, the molecule initially binds itself to the surface and only later is it interiorized by means of small vesicles that form in the flagellar pocket and in the final portion of the cytostome. The formed vesicles migrate to the median and posterior region of the cell, often forming tubular striations that are possibly the result of a fusion of the initial endocytical vesicles. Later, they merge with large structures, preferably located in the posterior region, giving origin to an organelle that contains electron-transparent inclusions that stand out within a matrix. Cytochemical studies indicate that the inclusions correspond to lipids, and the matrix consists essentiallly of proteins. All macro molecules ingested by the cell, regardless of their nature, are concentrated in this organelle, which is called a reservosome. It also contains cruzipain, a cysteine protease, the lysosomal hydrolase with the highest proteolytic activity in T. cruzi. In this organelle, this enzyme is active. In the lumen of reservosomes there is also a concentration of chagasin, a natural inhibitor of cruzipain, possibily responsible for the modulation of its activity. The pH inside the reservosome is about 6; therefore reservosomes, just like lysosomes in mammal cells, are an accumulation site for lysosomal hydrolases.Recent studies show that trypomastigotes and amastigotes have organelles that also contain the same lysosomal enzymes as reservosomes. Different experiments suggest that the organelles of trypomastigotes originate directly from the reservosomes of epimastigote forms, also containing non-digestible material (colloidal gold particles) previously endocytosed by the epimastigote forms, therefore showing that the organelles that contain lysosomal hydrolases of the trypomastigotes originate directly from the reservosomes of epimastigote forms.

Acidocalcisomes

For many years, the presence of cytoplasmatic structures has been described in T. cruzi and in other trypanosomatides. These structures are delimitated by a membrane unit and they contain an electrodense material which, in conventional preparations for electronic microscopy, occupies just part of the structure. These structures have been called volutin granules, dense bodies etc (Figure 11). The use of analytic electron microscopy indicates the presence of various elements, especially Ca²⁺ and P, in the organelle.Biochemical studies on T. cruzi and T. brucei show there is a cytoplasmatic organelle with acid pH, being acidified by the action of an ATPase proton of the vacuolar type, and which also has a Ca²⁺-ATPase. This structure was named acidocalcisome. Immunocytochemical studies using antibodies that recognized a vacuolar H+-ATPase of Dictiostelium and the Ca²⁺-ATPase of T. cruzi showed that acidocalcisomes basically correspond to electrodense granules. Electron microscopy studies show that the amastigote forms of T. cruzi have a much higher number of these structures than epimastigotes and trypomastigotes. This observation is very relevant, as the amastigote form lives in an intracellular environment in which the concentration of Ca²⁺ is normally low. Soon the parasite developed a special calcium accumulation mechanism in a specialized structure.

Contractile vacuole

Trypanosomatides have a set of tubules and vesicles located near the flagellar pocket, which form a structure known as contractile vacuole. It is involved in osmoregulation processes. In the case of epimastigote forms of T. cruzi , an association has been observed between the contractile vacuole and acidocalcisomes.

Nucleus

The nucleus of T. cruzi and of other trypanosomatides has a structural organization similiar to that of other eukaryotic cells. It is relatively small, measuring about 2.5 mm (Figure 13). In epimastigotes and amastigotes, the nucleus is slightly spherical. In trypomastigotes, it is elongated and located in the center of the cell.

The nuclear membrane has typical nuclear pores that are easily observed in cryofracture replicas. The pores have 80 nm of diameter, and there are about 25 pores per square micrometer of nuclear surface.In the interphase cell, the chromatin material agglomerates in masses located in the nuclear periphery (Figure 14), imediately beneath the nuclear membrane. Occasionally, chromatin masses project into the more central region of the nucleus. At the beginning of the cellular division, chromatin dispersion occurs, giving the nucleus a homogeneous appearance. The nucleolus, which is very evident, fragments and finally disappears.

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