5. Nuclear envelope

membrane and the PVs in a "kiss-and-flush"-like mechanism [22]. These factors are key components of the clathrin-dependent endocytic machinery in higher eukaryotes and protozoa

Figure 4. Schematic representation of the endocytic network of G. intestinalis. Active proteases reside primarily in the ER, where endocytosed proteins are degraded. PVs contain clathrin and are the site of initial uptake. Membrane fusions between PVs and between PVs and the ER are dynamic. Endocytosed proteins go from PVs to ER by dynamic fusions. ER,

Although electron microscopy revealed similar structures to endoplasmic reticulum (ER), there is still a controversy concerning the real presence of this organelle in Giardia. The cloning and characterization of SRα, a receptor for signal recognition particle (SRP) as well as the use of binding immunoglobulin protein (BiP), a ER resident 70 kDa heat shock protein, allowed the

alike.

92 Current Topics in Giardiasis

4. Endoplasmic reticulum

endoplasmic reticulum; PV, peripheral vesicles (from Abodeely et al. [19]).

One of the most intriguing features of G. intestinalis trophozoite is the presence of two nuclei with mirror symmetry (Figures 1, 2a and b, 5a). The nuclei are spherical or oval and symmetrically placed in the anterior portion of the cell (Figures 1, 2a and b, 8). Both nuclei are equivalent; they have the same amount of chromosomes, 2n = 10, and show a great homology when the nucleotide sequences are compared [30].

An inner and an outer membrane compose the nuclear envelope of higher eukaryote cells. The outer membrane is continuous with the ER membrane, which presents ribosomes engaged in protein synthesis. The inner nuclear membrane contains, in addition to the trilaminar membrane, filamentous proteins that form the nuclear lamina, which provides structural support for this membrane. The nuclear envelope of all eukaryotes is perforated by elaborated structures known as nuclear pore complexes [31].

The Giardia's nuclear envelope displays different profiles, such as blebs in the outer nuclear membrane envelope. This structure could correspond to the formation of vesicles from the endoplasmic reticulum that forms the outer nuclear membrane (Figure 5) [32]. Close proximity

Figure 5. Nuclei of G. intestinalis. Transmission electron microscopy (a, c and d) and freeze-fractured (figure b) images in non-encysting cells (a and b) and under process of encystation (c and d). (a) Both nuclei are similar in size; basal bodies and axonemes are just between the nuclei. (b) Pore complexes (arrowheads) similar in size and shape, with annular substructures. (c) In early stages of encystation (10 h) a nascent ESV (arrow) is close to nuclear envelope. (d) After 21 h of encystation the ESVs are closer to the peripheral vesicles (asterisks) and plasma membrane. N, nuclei; VD, ventral disc; ESV, encystation specific vesicle. (figure a: from Benchimol [35]; figure b: from Benchimol [34]; figures c and d: Midlej V, De Souza W and Benchimol, unpublished).

areas exist between the two nuclear membranes that become parallel but are distinct from the diaphragms found in nuclear envelopes of the eukaryotic cells [33]. Interestingly, the parasite pore complex distribution and clustering is different in each nucleus. Giardia nuclei are not identical; they seem to be either in different phases of chromosome condensation or they have different metabolic activity. Dividing nuclei displayed very few pore complexes, which is a characteristic of low metabolic activity and/or low nucleus-cytoplasm transport. The pore complexes in G. intestinalis are very uniform in size and shape, and they contain annular substructures (Figure 5b) that are similar to those of higher eukaryotic cells [33].

The parasite mitosis is not similar to other organisms, presenting different characteristics: (1) the nuclear envelope does not fragment completely during mitosis, leaving open places on the nuclei poles. This type of division is named semi-open mitosis, because only the nuclear poles are open. The spindle microtubules penetrate into the nuclei by these open poles. (2) Each nucleus moves to the central portion of the parasite, and one of them is located in the dorsal region and another in the ventral region and (3) the spindle is observed in the telophase [34]. Moreover, the parasite does not synchronize the nuclei division, and thus it is possible to find cells with three or four nuclei [35]. During the encystation process, the parasite mitosis still occurs; this is similar to what happens in the trophozoite vegetative form [36]. The nuclear division starts in the initial stages of encystation process through a semi-open mitosis. Bridges that originate by the nuclear membrane fusion connect the parental daughter nuclei. This interconnection between the nuclei remains intact while the parasite is in the cyst form; this is a characteristic of this stage in the Giardia life cycle [36].

Encysting cells show intranuclear inclusions that are morphologically similar to the ESVs and the ER membranes (Figure 5c and d), which may be a result of nuclear envelope folding. The presence of these inclusions could indicate intense ER activity since it forms from the outer nuclear membrane [33].
