However, only the members of ciliates and Euglena families develop the special cytostome-cytopharynx system. The answer is Yes. Paramecia have their way of excretion. After the nutrients from digested food have been absorbed into the cytoplasm, there is still indigestible debris inside the food vacuoles.
The waste will be ejected from a structure called the anal pore or cytoproct. Various single-celled eukaryotes have the anal pore.
The anal pore of a paramecium is a region of the pellicle that is not covered by ridges and cilia. The thin pellicle allows vacuoles to be merged into the cell surface and emptied. As we mentioned above, the outermost layer is the soft shell of pellicle and cilia. Bound to pellicle is a narrow peripheral layer of specialized firm cytoplasm, called the ectoplasm.
Below the ectoplasm lies a more fluid type of cytoplasm: the endoplasm. This region contains the majority of cell components and organelles. In this high-resolution image of the paramecium cell, you can see two layers of cytoplasm: ectoplasm and endoplasm. Trichocysts are protective organelles embedded in the ectoplasm layer.
Compared to the rest of the cytoplasm endoplasm , ectoplasm forms a thin, dense, and clear outer layer containing trichocysts and fibrillar structures. The roots of cilia also anchor in the ectoplasm layer. Pellicle and ectoplasm together serve as the protective skin for paramecia.
Trichocyst trick-o-sists is a small spindle-like organelle situated in the ectoplasm with a minute pore opened on the pellicle surface. Trichocysts are arranged perpendicular to the ectoplasm.
Trichocysts are filled with a dense refractive fluid containing swelled substances. When the cells receive mechanical, chemical, or electric stimuli, trichocysts discharge their contents and become long, thin, stinging spikes.
After they are discharged, new ones are generated from kinetosomes. The exact function of trichocysts is not quite clear, though a popular theory is that they are important for defense against predators. Trichocysts may also help cell adhesion and support the paramecium cell body. Trichocysts are spindle-like organelles that can discharge stinging filaments as a protection against predators.
Left: A TEM image showing a trichocyst embedded in the ectoplasm. When receiving outside stimuli, the core of trichocyst will swallow and push the spike out from the sheath.
Image: Bannister, J. Cell Sci. Right: Highly magnified phase contrast image showing a paramecium fired its spiky trichocysts for protection. Like a normal eukaryotic cell, enclosed inside the pellicle layer of paramecium is a jelly-like substance called cytoplasm.
The cytoplasm includes the cytosol and all the organelles. The cytosol is like condensed soup inside the cell. It is a complex mixture of all kinds of substances dissolved in water. You can find small molecules like ions sodium, potassium, or calcine , amino acids, nucleotides the basic units of DNA , lipids, sugars, and large macromolecules such as proteins and RNAs. Unlike the regular eukaryotic cells, paramecium has two nuclei , a big one and a small one.
Paramecium also consists of two types of vacuoles: contractile vacuole and food vacuole , which do not exist in human cells.
The most unusual characteristic of paramecia is their nuclei. They have two types of nuclei, which differ in their shape, content and function. White and black arrowheads point symbiotic bacteria inside the cytoplasm. Photo credit: MDPI. The two types of nuclei are micronucleus and macronucleus. The micronucleus contains all of the DNA called genome that is present in the organism. This DNA is passed from one generation to another generation during reproduction. On the other hand, the macronucleus contains a subset of DNA from the micronucleus.
These DNA fragments are copied from micronucleus to macronucleus because they carry genes that are frequently needed by the paramecium cell. Genes in the macronucleus are actively transcripted to mRNA and then translated to proteins.
The macronucleus is polyploid or contains multiple copies of each chromosome, sometimes up to copies. In other words, the function of the micronucleus is to maintain genetic stability and making sure that the desirable genes are passed to the next generation.
It is also called the germline or generative nucleus. Macronucleus plays a role in non-reproductive cell functions including the expression of genes needed for the everyday function of the cell. The macronucleus is also called the vegetative nucleus. The macronucleus acts as the random-access memory RAM which stores working data and machine codes. The computer only loads programs currently in use from hard drive to RAMs.
In a paramecium cell, more active genes meaning the cell need more of these proteins encoded by these genes may have more copies in the macronucleus. By having two nuclei, if a piece of DNA is in the micronucleus but not in the macronucleus, it will be removed during the next round of cell division.
In other words, if something foreign got into the micronuclear genome, then when the next macronucleus is made, it would be removed and not included in the expressed version [transcribed] of the genome. This mechanism functions as a primitive DNA immune system; that is, surveying the genome and trying to keep out invading elements.
Morphologically, macronucleus is kidney-liked or ellipsoidal in shape. The micronucleus is found close to the macronucleus. It is a small and compact structure, spherical in shape.
All paramecium species have one macronucleus. However, the number of micronuclei can vary by species. For example, P. Vacuoles take on specific functions in a paramecium cell. Classification 4. Cladistics 6: Human Physiology 1. Digestion 2. The Blood System 3. Disease Defences 4. Gas Exchange 5. Homeostasis Higher Level 7: Nucleic Acids 1. DNA Structure 2. Transcription 3. Translation 8: Metabolism 1. Metabolism 2. Cell Respiration 3. Photosynthesis 9: Plant Biology 1.
Xylem Transport 2. Phloem Transport 3. Plant Growth 4. Plant Reproduction Genetics 1. Meiosis 2. Plasma membrane and small vesicles inside the cell fluoresce a.
After a 10 min chase in unlabeled medium b fluorescence is visible in a few food vacuoles whereas after 30 min c small vesicles throughout the cytoplasm fluoresce. Bar, 20 m. Dextran-TXR, a fluid phase endocytosis marker, does not label the plasma membrane and enters the cell via small vesicles initially localized at the cortical level Figure 9a.
The vesicles later migrate in the cytoplasm and fuse with other endocytic vesicles and with food vacuoles Figure 9b. The number of labeled food vacuoles increases as the dextran-labeled vesicles fuse with food vacuoles Figure 9c and then decreases when the vacuolar content is digested and food vacuoles containing the indigestible material are ejected at the cytoproct.
Fusion of endocytic vesicles with food vacuoles. At first, green fluorescence is localized on plasma membrane a ; after 20 b and 30 min c of chase in unlabeled medium green fluorescence is also present inside the food vacuoles. Dextran internalization and intracellular flow. In cells labeled with dextran-TXR for 3 min fluorescence is visible in small vesicles located in the cortex under the plasma membrane a.
After a 10 min chase in unlabeled medium b , vesicles decrease in number and increase in size. After 30 min several food vacuoles are labeled c. The relationships between the two different routes of internalization, membrane transport and fluid phase endocytosis, are clearly shown when cells blocked in their phagocytic activity are simultaneously fed with WGA-FITC and dextran-TXR Figure The two probes probably partly join prior to their fusion with the phago-lysosomal compartment.
In order to understand if the two markers are internalized through two separate pathways, cells were incubated either in a hypertonic medium or in acetic acid. Indeed, subjecting mammalian cells to either incubation in media containing sucrose [ 49 ] or cytosol acidification with acetic acid [ 50 , 51 ] has been shown to inhibit clathrin-mediated endocytosis by interfering with clathrin-adaptor interactions [ 52 ], or by altering the structure of clathrin itself [ 53 - 55 ].
It also reduces dextran uptake, which is localized in small vesicles in the cortical part of the cell and in a few vesicles throughout the cytoplasm. Through cytosol acidification by 10 mM acetic acid, pH 5.
A similar fluorescent pattern was seen by using chlorpromazine data not shown , a cationic amphiphilic drug which inhibits clathrin-dependent receptor mediated endocytosis by reducing the number of coated pit-associated receptors at the cell surface [ 56 , 57 ]. Double labeling with WGA and dextran. Inhibition of clathrin-mediated endocytosis. Cells blocked in phagocytic activity are incubated in 0.
Sucrose inhibits WGA internalization and reduces dextran internalization. Cells blocked in phagocytic activity are incubated in 10 mM acetic acid, pH 5. Green fluorescence is localized on the plasma membrane and red fluorescence in vesicles in both the cortical region and throughout the cytoplasm. Bars, 20 m. Conversely, dextran internalization is blocked by filipin and nystatin Figure 12 , sterol-binding agents that disrupt caveolar structure and function [ 58 ]. Inhibition of clathrin-independent endocytosis.
Effect of nystatin on fluid phase and membrane mediated transport. Green fluorescence locate on the plasma membrane and in vesicles through the cytoplasm; no red vesicles are seen inside the cell. The classical paradigm of receptor function assumes that receptors localize on the cell surface and are activated by the binding of agonist ligands. After activation, most receptors are endocytosed from cell surface and travel to low pH endosomes, allowing the ligand to detach before the receptor is recycled back to the cell surface or sent through late endosomes to lysosomes for degradation [ 59 ].
Increasing evidence shows that some G protein-coupled receptors are not totally inactive in the absence of ligands but exhibit a constitutive activity, too, with elevated basal levels of intracellular signaling [ 60 , 61 ]. It was found that receptor internalization from the neuronal surface occurring both constitutively and in response to agonist exposure is mediated by clathrin-dependent endocytosis [ 62 - 64 ].
Clathrin-coated vesicles are the initial vehicles for sequestration of surface receptors, which are ultimately degraded or recycled. Endocytosis of such membrane proteins involves a series of steps beginning with the clustering of receptors at specific sites of the plasma membrane, regions that later turn into clathrin-coated pits. Receptors do this by recruiting cytosolic AP2 adaptor complexes through their cytoplasmic tails.
AP2 is a key component of the endocytic machinery that links cargo membrane proteins to the clathrin lattice, selects molecules for sorting into clathrin-coated vesicles and recruits clathrin to the plasma membrane [ 65 - 69 ]. In addition to the AP2 adaptor complex, amphiphysin interacts with dynamin and the disruption of dynamin-amphiphysin interaction by recombinant amphiphysin src homology 3 SH3 domain in vivo leads to a potent block in clathrin-mediated endocytosis [ 70 , 71 ].
Dynamin, a large GTP-binding protein, pinches off vesicles at constricted clathrin-coated pits by forming a ring-like structure collaring the neck of the vesicle that is thought to drive vesicle separation. Eps15 function in clathrin-dependent endocytosis seems to be restricted to the early events leading to clathrin-coated pit formation: indeed eps15 is not present in clathrin-coated vesicles [ 72 ]. It has been shown that endocytosis of receptors may also occur through other membrane structures, including noncoated membrane invaginations [ 73 , 74 ] and caveolae [ 75 ].
The 2 -adrenergic receptor, which is endocytosed by clathrin-coated pits in several cell types [ 76 , 77 ], is endocytosed by membrane invaginations resembling to caveolae in other cells [ 74 , 75 ]. Cholecystokinin receptors have been observed in both clathrin-coated pits and caveolae in the same cells [ 73 ]. Caveolae are cholesterol- and sphingolipid-rich smooth invaginations of the plasma membrane that partition into raft fractions and the expression of which is associated with caveolin 1.
An homologue of dynamin, a protein present in mammalian cells with three isoforms generating more than 25 possible spliced variants expressed in a tissue-specific manner, was identified in Paramecium , too [ 80 ]. Endocytosis in Tetrahymena also involves a protein in the dynamin family [ 82 ]. Endocytosis of receptors can contribute to functional resensitization of signal transduction by promoting dephosphorylation and recycling of receptors to the plasma membrane [ 83 ] as well as to down-regulation of receptors, a process that leads to functional desensitization of signal transduction by reducing the number of receptors present in the plasma membrane and promoting degradation of receptors in lysosomes [ 73 , 84 , 85 ].
These processes of receptor regulation are thought to involve membrane trafficking of receptors via distinct recycling or degradative pathways and can mediate opposite effects on the regulation of functional signal transduction [ 83 , 86 ]. Golgi-derived vesicles provide newly synthesized receptors to the cell surface, whereas clathrin coated vesicles are the initial vehicles for sequestration of surface receptors, which are ultimately degraded or recycled back to the plasma membrane, either directly or through the recycling endosomes [ 87 - 89 ].
These processes are mediated by a continuous traffic of vesicular and tubular intermediates which needs to be coordinated to ensure proper progression of cargo through the different compartments.
Several rab family members have been localized to distinct compartments of the endocytic pathway and play different roles in endocytosis and recycling [ 90 - 93 ]. Rab5 and rab4 are both localized to early endosomes but exert opposite effects on the uptake of membrane-bound proteins. Rab5 plays a role in the formation of clathrin-coated vesicles at the plasma membrane [ 94 ], their subsequent fusion with early endosomes, in the homotypic fusion between early endosomes [ 95 , 96 ] and in the interaction of early endosomes with microtubules [ 97 ].
Rab4 has been implicated in the regulation of membrane recycling from the early endosomes to the recycling endosomes or directly to the plasma membrane [ 98 ]. In accordance with this functional diversity, rab5 lies at the center of a complex machinery comprising several effector proteins [ 99 ]. EEA1 is predominantly localized to the early endosomes and is regarded as a specific marker of this compartment. Because of this localization and given its function in endosome membrane docking [ 99 ] it has been proposed that EEA1 may confer directionality to rab5-dependent vesicular transport to the early endosomes.
Another effector protein for rab5 is rabaptin Rabaptin-5 binds directly to the GTP-bound form of rab5 and is recruited to early endosomes by rab5 in a GTP-dependent manner [ ], stabilizes rab5 in the GTP-bound active form by down-regulating GTP hydrolysis [ ] and, finally, it is required for the homotypic fusion between early endosomes as well as for the heterotypic fusion of clathrin-coated vesicles with early endosomes in vitro [ , ].
Rabadpin-5 also interacts, via a distinct structural unrelated N-terminal RBD, with GTP-bound rab4 but does not appear to interact with rab11, a GTPase that is highly enriched on the recycling endosome and whose activity is required for receptor recycling through this compartment [ 89 ].
Thus the same effector interacts with the two rab proteins which act sequentially in transport through the early endosomes. Furthermore, the lysosomal sorting of receptors is dependent upon rab 7 activity [ ]. Small GTPase rab is a widely conserved molecular switch among eukaryotes and regulates membrane trafficking, also in ciliates.
In the T. These do not include 17 putative rabs previously reported [ 82 ]. This is a remarkable number, considering that somewhat over 63 rabs have been identified in humans [ ]. Some of them are very conserved and some others are ciliate specific [ , ].
Endocytic compartments were found to be associated with a large number of rabs, including both conserved endocytic rabs but also a roughly equal number of divergent rabs. One of the conserved rabs did not fall into any of the proposed core clades. The animal rabs in this clade are associated with transport of lysosome-related organelles, while the Tetrahymena protein localized to phagosomes.
By comparing the amino acid sequence of rabs in humans and the budding yeast Saccharomyces cerevisiae , 42 conventional and 44 species-specific rabs were categorized in Tetrahymena and conventional and 72 species-specific rabs in Paramecium.
Among them, nine Paramecium rab genes showed high homology to seven Tetrahymena rabs, suggesting the conservation of ciliate-specific rab [ ]. In our studies we are interested in understanding the endocytic properties of GABA B receptors in Paramecium [ , ]. Although most G protein-coupled receptors undergo endocytosis, the conditions and mechanisms of this process vary from receptor to receptor.
Many of them are endocytosed via clathrin-coated pits, but some are not [ , ]. Some have an agonist-induced endocytosis, some are continuously endocytosed even in the absence of stimulation, while others exhibit both a constitutive and a stimulated endocytosis [ , , ]. Currently, very little is known about the targeting and trafficking mechanisms of GABA B receptor in cells.
It has been shown that GABA A receptors are internalized by a clathrin-coated pit-mediated process in hippocampal neurons and in A cells [ ] and in a clathrin independent manner in HEK cells [ ].
Using a dominant-negative dynamin construct K44A Herring et al. It was also shown that both recombinant and neuronal GABA A receptors can constitutively recycle between the cell surface and an intracellular endosomal compartment [ ]. In Paramecium a dynamin- and clathrin-dependent pathway has been already observed [ 78 , 79 ]. Constitutive internalization and intracellular trafficking of receptors in P.
GABA B receptors display a dotted vesicular pattern dispersed on the cell surface and throughout the cytoplasm Figure 13a , and are internalized via clathrin-dependent and -independent endocytosis. Indeed, GABA B receptors colocalize with the adaptin complex AP2, which is implicated in the selective recruitment of integral membrane proteins to clathrin-coated vesicles, and with caveolin 1, which is associated with uncoated membrane invaginations [ ]. Cells were double labeled with a guinea pig anti-GABA B receptor R1 subunit antibody and with a monoclonal anti-clathrin or anti-caveolin 1 antibody and visualized with Alexa Fluor conjugated anti-guinea pig and Alexa Fluor conjugated anti-mouse secondary antibodies, respectively.
Staining with anti-clathrin or antI-caveolin antibody led to a punctuate pattern throughout the cytoplasm representing endocytic vesicles. The expression of GABA B receptors and clathrin- or caveolin-coated vesicles exhibited a clustered distribution on the cell membrane and inside the cytoplasm Figure Importantly, GABA B receptor and clathrin- or caveolin-coated vesicle clusters were partly colocalized yellow fluorescence.
In cells labeled with a polyclonal antibody against GABA B receptor b and a monoclonal antibody against clathrin HC a , a clustered distribution of fluorescence is detected on the plasma membrane and inside the cytoplasm.
GABA B receptors and clathrin vesicles are partly colocalized c, yellow fluorescence. In addition, we have shown that GABA B receptors are removed from the plasma membrane by clathrin-dependent and -independent endocytosis by blocking receptors internalization by hypertonic sucrose. However, it has recently been found that sucrose inhibits GABA A receptor endocytosis that is not mediated by clathrin-coated pits [ ].
Therefore, we have also used cytosol acidification with acetic acid for clathrin-mediated endocytosis inhibition [ 50 ]. Furthermore, GABA B receptor internalization in Paramecium is blocked by filipin and nystatin, cholesterol binding drugs. The sensitivity of endocytosis to nonacute cholesterol depletion with agents such as filipin and nystatin distinguishes caveolae and raft pathways from clathrin-dependent and constitutive pinocytosis pathways [ ].
Treatment of cells with mM sucrose or cytosol acidification significantly inhibited the internalization of receptors, as shown by the considerable reduction in receptors inside the cytoplasm Figure 14 as compared to the control Figure14b.
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