EUKARYOTE

I. Eukaryotic cells - Can be unicellular (protists such as yeast, paramecium and amoebae) or multicellular (a system of division of labor such as fungi, animals, and plants).

A. First appeared 1.5 billion years ago.

B. Distinguishing characteristics (in comparison to prokaryotes)..

1. Generally larger than prokaryotic cells - generally 20 -> 50 micrometers, but can even be up to meters in length.

2. Eukaryotic DNA works in much the same way as prokaryotic DNA and the same code is used, however there are some distinct differences as well:

a) Eukaryotic DNA is enclosed by a membrane making a well defined nucleus.

b) Eukaryotic DNA tends to have protein bound to it forming chromatin (chromosomes). The regulating proteins (also found in prokaryotes) turn on and off genes. The histones (in larger mass but less important) help fold eukaryotic DNA.

c) The DNA has repeated and 'junk' areas (e.g. introns).

d) The DNA has free ends and is not bound to any membranes.

e) Replication starts at several points.

3. The nucleus containerizes the DNA.

a) Nuclear structure of the membrane.

(1) Double membrane

(2) This double membrane is apparently derived from or closely associated with the rough endoplasmic reticulum (r.e.r).

(a) Transmission electron microscopy and radioactive flow studies suggest continuity with the r.e.r..

(b) Functional ribosomes occur on the outside of the nuclear membrane but not on the inside (remember that ribosomes are probably binding and unbinding all of the time and there are no ribosomes inside the nucleus to bind)

(c) It has been shown that proteins derived from these ribosomes on the 'nuclear membrane' can be channeled through the inner membrane space of the r.e.r. , then smooth e.r. and end up in the Golgi apparatus.

(d) During mitosis and meiosis the nuclear membrane seems to disappear but probably just unfolds to become regular r.e.r.; and then apparently reforms afterwards from the endoplasmic reticulum.

(3) The outer structure of the nucleus actually seems to come from a strong structural protein layer called the nuclear lamina. Then the r.e.r. simply folds around this already existing structural protein surface.

b) The transport properties of the nuclear membrane are poorly understood.

(1) There is apparently an ATPase used for active transport.

(2) 'Nuclear pores'

(a) a. Gold particles (presumably inert) up to 10 nm can easily diffuse into the nuclei, suggesting the pores are just that: open gaps. However, other evidence suggests this is too simplistic of a concept.

(b) Some ions and some other small molecules can not diffuse in easily. Nuclei also have osmotic properties suggesting a semipermeable membrane.

(d) The membrane is some times infolded in metabolically active cells. This would suggest the larger surface area is for increased transport function (or membrane associated reactions).

c) Chromatin - The combination of DNA and protein forming stranded material. In a nondividing cell the chromatin seems disperse (but often near the membrane) in transmission electron microscopy (the chromosomes are just highly unfolded). Such material "condenses" during mitosis at which time they can be seen with staining and a light microscope (this is the way we normally visualize chromosomes).

d) Nucleolus - is the location on the DNA in the nucleus where ribosomal RNA is made. The pre-ribosomes assemble and 'hang around' the DNA while they get processed.

(1) All eukaryotes have at least one nucleolus and some may have more depending upon the species.

(2) Although prokaryotes also must make ribosomal RNA, it does not stay near the DNA after being made, hence prokaryotes have no visible nucleolus.

e) Why might it be important for the cell to have a nuclear compartment?

(1) Provides an ideal environment for the transcription reactions (The cytoplasm, in essence, becomes a compartment too and can specialize in translation reactions)

(2) Provides an ideal environment for the replication reactions.

(3) Separates transcription from translation thus allowing for m-RNA processing.

f) How important is the nucleus to the life of a cell?

(1) No eukaryotic cell has ever been found where a nucleus does not exist at some point of its life cycle.

(2) A mature human red blood cell exists and functions ( although a narrow function) for about 120 days without a nucleus.

(3) A sea urchin egg can undergo cell division (although not growth) several times with its nucleus artificially removed.

(4) Acetabularia is a large, single celled alga with a single localized nucleus. Its nucleus can be easily cut out. When this is done, the cell can live (i.e. do photosynthesis, respiration, protein synthesis etc.) up to a year.

(5) A cell can still carry out protein synthesis as long as its m-RNA stays intact. The turnover rate for m-RNA varies greatly depending upon the protein and the cell. Acetabularia has long lasting m-RNA. Higher cells tend to have higher turnover rates, but this allows for more control of what is happening in higher cells.

(6) Even if the m-RNA is not there, life may continue depending upon how long the enzymes last (remember it is the enzymes that control the reactions of life). The turnover rates for enzymes also varies greatly from protein to protein and cell to cell.

4. Membrane bound organelles besides the nucleus.

a) Endoplasmic reticulum (e.r.) Tubular/lamellar membrane system with a space between the membranes (lumen).

(1) Rough e.r. has ribosomes while smooth e.r. does not. Rough e.r has "attached" (transient might be a better term) ribosomes. When those attached ribosomes make a protein, they deposit it into the lumen of the rough e.r. The protein may be processed in the lumen area. It may also be transported in the lumen space to other parts of the cell. It may even pass into the smooth e.r (no ribosomes) which has a physical connection with the rough e.r.

(2) Endoplasmic reticulum has the ability to assemble the lipids needed in making membranes. Therefore, e.r. is thought to make its own membrane.

(3) E.r. is important in certain types of reactions (protein processing, lipid formation, membrane formation and detoxifying reactions).

(4) E.r. pinches off to form microbodies.

(5) Rough e.r. is thought to be continuous with the nuclear membrane.

(6) Smooth e.r. forms the Golgi apparatus.

b) Endomembrane system: It is thought that all of the membranes of a eukaryotic cell are initially formed by the endoplasmic reticulum, except for the chloroplast and mitochondria. The nuclear membrane comes from e.r., the e.r. membrane is formed by e.r., microbodies come from e.r., Golgi comes from e.r., all organelle membranes (except mitochondria and chloroplasts) come from Golgi which got it from e.r., and there is evidence that the cell membrane itself comes from secretory vesicles arising from Golgi which arose from e.r.. Mitochondria and chloroplasts seem to divide on their own (see theory of endosymbiosis) and are the only membranes not derived from e.r., hence the only membranes not part of the endomembrane system.

c) Dictysome membranes making up the Golgi apparatus

(1) Golgi packages proteins and protein related compounds into secretory vesicles and/or certain other organelles (e.g. lysosomes, vacuoles).

(2) Golgi also carries out certain reactions allowing for the formation of certain important macromolecules (particularly important in adding sugars to proteins). Made of stacks of membranes from smooth e.r..

d) Lysosomes: contain enzymes for breaking down un-needed macromolecules into basic reusable components. A vesicle with a single membrane, about 0.7 µm in diameter. Its malfunction is involved in several important diseases including Tay-Sachs disease.

e) Mitochondria: convert certain kinds of chemical energy to ATP chemical energy. Double membrane with the inner membrane highly infolded. Not part of the endomembrane system.

f) Vacuoles and vesicles: single membrane bound compartments where liquid, fatty, or solid matter is stored. Enclosed by a single membrane. The membrane of a vacuole is a tonoplast. The term vesicle is usually used for such a structure when it is smaller, more transitory, and/or found in an animal cell.

g) Microbodies (peroxisomes): contain enzymes for certain oxidation reactions.

5. Ribosomes are larger and distinct from the ribosomes found in prokaryotes. The nucleotide sequence is different. They are both free in the cytoplasm and membrane bound to endoplasmic reticulum. They, themselves have no membranes - rather they can be regarded as a clump of nucleic acid and protein.

6. Microtubules, microfilaments (actin filaments) and intermediate filaments. (some form of these may also exist in prokaryotes) are filamentous proteins with the capability of changing shape by using ATP energy. Used for cell shape, movement, flagella, cilia, centrioles, spindle apparatus, etc. (No membrane).

a) Microscopy of microtubules and microfilaments.

(1) Classically the heavy metal of OsO4 (osmium tetroxide) has been used to stain cells for transmission scanning electron microscopy in order to get nice clear images. Unfortunately the reason the pictures are clear is that microtubules and microfilaments are dissolved by the OsO4 stain! Thus, for many years the importance of microtubules and microfilaments have been overlooked.

(2) New stains clearly show microtubules and microfilaments in electron micrographs. It is very common to find microtubules and microfilaments near the cell membrane for the apparent purpose of giving the cell shape and structure.

(3) A new technique of using fluorescent tagged antibodies against tubulin allow you to see microtubules and microfilaments with a regular microscope as brilliantly as if a Christmas tree were lit up.

b) Microfilaments are made of spherical actin protein molecules strung together in a long filamentous chain called a microfilament. They are used for giving a cell shape and structure, probably involved in cytoplasmic streaming, and probably involved in directed movement of organelles. Myosin and some other proteins also seems to be involved. (note that actin and myosin are important components of muscle and that the contractive feature of muscles is obviously highly evolved microfilaments.

5-7 nm in diameter

(1) Cytoskeleton - give animal cells shape. Cytochalasin causes cells to round up into balls; the shape that minimizes surface tension.(Note that actin is also found in plant cells).

(2) Cell shape changes:

(a) Ameboid movement (not only done by amoeba but by many cells. When a new pseudopod forms, it is done forming new microfilaments into the new pseudopod.

(b) Cytokinesis in animal cells

(3) Development and growth

(4) Cytoplasmic streaming

(5) Exocytosis and endocytosis

(6) Membrane protein manipulation

(7) Possible mechanisms: Sliding mechanism and/or assembly - disassembly. For both ameboid movement and cytokinesis the mechanism seems to be assembly-disassembly.

(8) Control often seems to involve Ca++; high calcium often seems to cause depolymerization.

(9) The proteins (actin and myosin) that make up muscles of animals is very similar to that found in microfilaments. Apparently muscle cells are cells that have become highly specialized with respect to microfilaments, thus allowing for their magnificent contractile qualities! Note that ATP is used for muscle contraction, Ca⁺⁺ is used for control of contraction, and the sliding filament model is the one used for muscle contraction!

c) Microtubules are made of tubulin protein molecules (plus other proteins such as myosin) in to long chains and then 13 such chains line up next to each other to form a tube. These can even associate into higher level structures such as centrioles, flagella, and cilia. Microtubules are what makes up the spindle apparatus during mitosis and meiosis, they are the moving parts of cilia and flagella, they also make up centrioles and basal bodies.

colchicine is a drug that can be used to artificially break down microtubules.

(1) Spindle apparatus is made of microtubules. The structure that pulls chromosomes into two new cells.

(a) Some tubules go from pole to pole and lengthen during mitosis and meiosis.

(b) Other microtubules strands go from the chromosomal centromere to the pole and shorten.

(d) Control mechanisms of this process are unknown.

(2) Centrioles

(a) Often found as a perpendicular pair near the nucleus.

(b) Nine triplets of microtubules as seen in a cross sectional view.

(c) Centrioles are very closely analogous to basal bodies and there is evidence that basal bodies develop from centrioles.

(d) Not found in higher plant cells and certain other cell types! Flagella and cilia also tend not to be found in higher plant cells.

(e) Are apparently involved in the very initial stages of spindle formation..

(3) Basal bodies - similar in structure to centrioles with 9 triplet tubules and are presumably made from centrioles. Always found near the cell membrane and they seem to lead to the formation of cilia and flagella.

(4) Cilia and flagella - both have the same general structure and function, it is just in the case of cilia that there is a lot more of them. (cilia are thought of as being short and numerous per cell, flagella are thought of as being long and only one or two per cell.)

(a) 9 + 2 structure of microtubules (tubulin) with spokes from the inner two to the outer nine. The cross bridges between the outer tubules are the energy users called dynein. This dynein can use ATP that leads to movement as described below.

(b) They can be removed from a cell intact (using a laser beam) yet they still function! Even the membrane can be removed.

(c) The theory of how these work is that the outer tubules will slide with respect to the adjacent outer tubules (i.e. sliding filaments) using the dynein as the ATPase.

(d) Cilia and flagella, for some unknown reason, seem to have some inherent sensory function with the cellular plasma membrane that covers the filaments.

(5) cytoskeleton

d) Intermediate filaments.

II. Distinguishing characteristics of eukaryotic plant and animal cells.

A. Plant cell Þ a plant cell either directly uses light energy or is part of an organism that does (or has in the evolutionary past used light energy).

a) Cell wall of cellulose

b) Usually has plastids (chloroplasts are the most prominent of these and are present in green parts of the plant.; we will see leucoplasts in lab)

c) Centrioles absent (?)

d) Tend to have large vacuoles.

e) In multicellular systems, the cells tend to be continuous with cytoplasmic connections (plasmodesmata).

2. Animal cells, Fungi and Protists Þ completely dependent upon chemical energy.

a) Never have a cell wall (except fungi)

b) Never have plastids

c) usually have centrioles

ENDOSYMBIOTIC THEORY

I. Theory of endosymbiosis.

A. Introduction

1. Simply stated, the theory of endosymbiosis is the concept that mitochondria and chloroplasts are the result of years of evolution initiated by the endocytosis of bacteria and blue-green algae which, instead of becoming digested, became symbiotic.

2. Alternate theories of the evolution of eukaryotic cells.

a) The mitochondria and chloroplasts are developments of the invaginations of the cell membrane. Such that the rest of the cell, being relieved from power house duties, became highly developed and evolved.

b) The bacterium some how developed externally, and the casing around the bacterium is now what we call a eukaryotic cell.

3. We will never be able to turn back the clocks, thus we will never be absolutely sure of the correct answer.

B. Is it feasible to consider that a bacterium was once ingested by a "eukaryotic cell" such that the bacterium became symbiotic?

1. Some dinoflagellates have been observed to have two nuclei. It is now known that one alga cell is completely encompassed within the cell of the dinoflagellate. They apparently undergo simultaneous mitosis and cellular division.

2. Viable chloroplasts can be incorporated (from algae) into the gut cells of a sea slug. These chloroplasts seem to be non-dividing, but survive much longer than we can get chloroplasts to survive in vitro.

3. Mouse fibroblast cells will artificially take up isolated chloroplasts, and the latter will remain functional for a time. The same can be done for mitochondria.

4. There are ciliated protozoa with chloroplasts and eye spots.

5. There are obvious possible ecological values for endosymbiotic relationships.

C. Factors in favor of mitochondrial and chloroplast endosymbiosis.

1. Outer membrane is similar to the plasma membrane.

2. Mitochondria look a lot like bacteria; chloroplasts look a lot like blue-green algae. These organelles are similar to prokaryotes in that:

a) Both have circular, naked (little protein) DNA. Most other quirks that distinguish prokaryotic DNA and eukaryotic DNA (except introns), suggest mitochondrial and chloroplast DNA is prokaryotic.

b) RNA is similar

c) Both have prokaryotic type and size ribosomes.

d) Inner membrane lipids of mitochondria and chloroplasts are similar to their prokaryotic counterparts.

e) Membrane proteins are highly similar (All of the electron transport enzymes and the H+ ATPase - these are NEVER found any where else in the eukaryotic cell other than mitochondria and/or chloroplasts.)

f) Soluble enzymes are very similar (e.g. Krebπs cycle enzymes).

3. Mitochondria and chloroplasts seem to divide independently of the rest of the eukaryotic cell.

4. Eukaryotes are very good at endocytosis.

D. Factors against the theory:

1. Mitochondria and chloroplasts only code for a few proteins. Most of the proteins found in the organelles are actually coded for by the nuclear DNA. (Did the organelle DNA jump to the nuclear DNA in evolutionary history?)

a) Because of this, it is not surprising that mitochondria nor chloroplasts have been shown to be able to live on their own when isolated from the eukaryotic cell.

2. Mitochondrial and chloroplast DNA have introns, a phenomenon never seen in prokaryotes.(Did this characteristic jump from the nuclear DNA to the organelle DNA?)

3. If the theory of endosymbiosis is true, then one must ask what was the original eukaryotic cell (without mitochondria or chloroplasts) and how did it survive (glycolysis?). Why have not any primitive eukaryotic cells ever be found that are devoid of these organelles (is today's eukaryote just too superior?)