Several independent lines of evidence suggest that the
mitochondria and chloroplasts of modern eukaryotes were derived during evolution
from aerobic bacteria and cyanobacteria that took up endosymbiotic residence in
early eukaryotic cells (Fig. 2-17; see also Fig. 2-7). Mitochondria are always
derived from preexisting mitochondria, and chloroplasts from chloroplasts, by
simple fission, just as bacteria multiply by fission. Mitochondria and
chloroplasts are in fact semiautonomous; they contain DNA, ribosomes, and the
enzymatic machinery to synthesize proteins encoded in their DNA. Sequences in
mitochondrial DNA are strikingly similar to sequences in certain aerobic
bacteria, and chloroplast DNA shows strong sequence homology with the DNA of
certain cyanobacteria. The ribosomes found in mitochondria and chloroplasts are
more similar in size, overall structure, and ribosomal RNA sequences to those of
bacteria than to those in the cytoplasm of the eukaryotic cell. The enzymes that
catalyze protein synthesis in these organelles also resemble those of the
bacteria more closely.
If mitochondria and chloroplasts are the descendants of early bacterial
endosymbionts, some of the genes present in the original freeliving bacteria
must have been transferred into the nuclear DNA of the host eukaryote over the
course of evolution. Neither mitochondria nor chloroplasts contain all of the
genes necessary to specify all of their proteins. Most of the proteins of both
organelles are encoded in nuclear genes, translated on cytoplasmic ribosomes,
and subsequently imported into the organelles.
Several types of protein filaments visible with the electron microscope
crisscross the eukaryotic cell, forming an interlocking three-dimensional
meshwork throughout the cytoplasm, the cytoskeleton. There are
three general types of cytoplasmic filaments: actin filaments, microtubules, and
intermediate filaments (Fig. 2-18). They differ in width (from about 6 to 22
nm), composition, and specific function, but all apparently provide structure
and organization to the cytoplasm and shape to the cell. Actin filaments and
microtubules also help to produce the motion of organelles or of the whole
cell.
Each of the cytoskeletal components is composed of simple protein subunits
that polymerize to form filaments of uniform thickness. These filaments are not
permanent structures; they undergo constant disassembly into their monomeric
subunits and reassembly into filaments. Their locations in cells are not rigidly
fixed, but may change dramatically with mitosis, cytokinesis, or changes in cell
shape. All types of filaments associate with other proteins that cross-link
filaments to themselves or to other filaments, influence assembly or
disassembly, or move cytoplasmic organelles along the filaments.
Actin is a protein present in virtually all eukaryotes, from the protists to
the vertebrates. In the presence of ATP, the monomeric protein spontaneously
associates into linear, helical polymers, 6 to 7 nm in diameter, called actin
filaments or microfilaments (Fig. 2-19).
The importance of actin polymerization and depolymerization is clear from the
effects of cytochalasins, compounds that bind to actin and block polymerization.
Cells treated with a cytochalasin lose actin filaments and their ability to
carry out cytokinesis, phagocytosis, and amoeboid movement. However, chromatid
separation at mitosis is not affected, ruling out an essential role for actin in
this process. Compounds such as cytochalasins, which are naturally occurring
poisons or specific toxins, are often very helpful in experimental studies in
pinpointing the important participants in a biological process.
Cells contain proteins that bind to actin monomers or filaments and
influence' the state of actin aggregation (Fig. 2-19). Filamin and fodrin
cross-link actin filaments to each other, stabilizing the meshwork and greatly
increasing the viscosity of the medium in which the filaments are suspended; a
concentrated solution of actin in the presence of filamin is a gel too viscous
to pour. Large numbers of actin filaments bound to specific plasma membrane
proteins lie just beneath and more or less parallel to the plasma membrane,
conferring shape and rigidity on the cell surface.
Myosins Move along Actin Filaments Using the Energy of ATP
Actin filaments bind to a family of proteins called myosins,
enzymes that use the energy of ATP breakdown to move themselves along the actin
filament in one direction. The simplest members of this family, such as myosin
I, have a globular head and a short tail (Fig. 2-20). The head binds to and
moves along an actin filament, driven by the breakdown of ATP. The tail region
binds to the membrane of a cytoplasmic organelle, dragging the organelle behind
as the myosin head moves along the actin filament. It appears likely that
myosins of this type bind to various organelles, providing specific transport
systems to move each type of organelle through the cytoplasm. This motion is
readily seen in living cells such as the giant green alga Nitella;
endoplasmic reticulum, as well as mitochondria, nucleus, and other
membrane-bound organelles and vesicles, move uniformly around the cell at 50 to
75 μm/s in a process called cytoplasmic streaming (Fig. 2-20). This motion has
the effect of mixing the cytoplasmic contents of the enormous algal cell much
more efficiently than would occur by diffusion alone.
A larger form of myosin is found in muscle cells, and also in the cytoplasm
of many nonmuscle cells. This type of myosin also has a globular head that binds
to and moves along actin filaments in an ATP-driven reaction, but it has a
longer tail, which permits myosin molecules to associate side by side to form
thick filaments (see Fig. 7-31). Contractile systems composed of actin and
myosin occur in a wide variety of organisms, from slime molds to humans.
Actin-myosin complexes form the contractile ring that squeezes the cytoplasm in
two during cytokinesis in all eukaryotes. In multicellular animals, muscle cells
are filled with highly organized arrays of actin (thin) filaments and myosin
(thick) filaments, which produce a coordinated contractile force by ATP-driven
sliding of actin filaments past stationary myosin filaments.
Microtubules Are Rigid, Hollow Rods Composed of Tubulin Subunits
Like actin filaments, microtubules form spontaneously from their monomeric
subunits, but the polymeric structure of microtubules is slightly more complex.
Dimers of α- and β-tubulin form linear polymers (protofilaments), 13 of which
associate side by side to form the hollow microtubule, about 22 nm in diameter
(Fig. 2-21). Most microtubules undergo continual polymerization and
depolymerization in cells by addition of tubulin subunits primarily at one end
and dissociation at the other. Microtubules are present throughout the
cytoplasm, but are concentrated in specific regions at certain times. For
example, when sister chromatids move to opposite poles of a dividing cell during
mitosis, a highly organized array of microtubules (the mitotic spindle; Fig.
2-14) provides the framework and probably the motive force for the separation of
chromatids. Colchicine, a poisonous alkaloid from meadow saffron, prevents
tubulin polymerization. Colchicine treatment reversibly blocks the movement of
chromatids during mitosis, demonstrating that microtubules are required for this
process.
Microtubules, like actin filaments, associate with a variety of proteins that
move along them, form cross-bridges, or influence their state of polymerization.
Kinesin and cytoplasmic dynein, proteins found in the cytoplasm of many cells,
bind to microtubules and move along them using the energy of ATP to drive their
motion (Fig. 2-22). Each protein is capable of associating with specific
organelles and pulling them along the microtubule over long distances at rates
of about 1μm/s. The beating motion of cilia and eukaryotic flagella also
involves dynein and microtubules
The Motion of Cilia and Flagella Results from Movement of Dynein along
Microtubules
Cilia and flagella, motile
structures extending from the surface of many protists and certain cells of
animals and plants, are all constructed on the same microtubule-based
architectural plan (Fig. 2-23). (Although they bear the same name, the flagella
of bacteria (p. 28) are completely different in structure and in action from the
flagella of eukaryotes.) Eukaryotic cilia and flagella, which are sheathed in an
extension of the plasma membrane, contain nine fused pairs of microtubules
arranged around two central microtubules (the 9 + 2 arrangement; Fig. 2-23).
Ciliary and flagellar motion results from the coordinated sliding of outer
doublet microtubules relative to their neighbors, driven by ATP. The motions of
cilia and flagella propel protists through their surrounding medium, in search
of food, or light, or some condition essential to their survival. Sperm are also
propelled by flagellar beating. Ciliated cells in tissues such as the trachea
and oviduct move extracellular fluids past the surface of the ciliated tissue.
The contraction of skeletal muscle, the propelling action of cilia and
flagella, and the intracellular transport of organelles all rely on the same
fundamental mechanism: the splitting of ATP by proteins such as kinesin, myosin,
and dynein drives sliding motion along microfilaments or microtubules.
The third type of cytoplasmic filament is a family of structures with
dimensions (diameter 8 to 10 nm) intermediate between actin filaments and
microtubules. Several different types of monomeric protein subunits form
intermediate filaments. Some cells contain large amounts of one
type; some types of intermediate filament are absent from certain cells; and
some cell types apparently lack intermediate filaments altogether. As is the
case for actin filaments and microtubules, intermediate filament formation is
reversible, and the cytoplasmic distribution of these structures is subject to
regulated changes.
The function of intermediate filaments is probably to provide internal
mechanical support for the cell and to position its organelles. Vimentin
(Mr 57,000) is the monomeric subunit of the
intermediate filaments found in the endothelial cells that line blood vessels,
and in adipocytes (fat cells). Vimentin fibers appear to anchor the nucleus and
fat droplets in specific cellular locations. Intermediate filaments composed of
desmin (Mr 55,000) hold the Z disks of striated muscle
tissue in place. Neurofilaments are constructed of three different protein
subunits (Mr 70,000, 150,000, and 210,000), and
provide rigidity to the long extensions (axons) of neurons. In the glial cells
that surround neurons, intermediate filaments are constructed from glial
fibrillary acidic protein (Mr 50,000).
The intermediate filaments composed of keratins, a family of structural
proteins, are particularly prominent in certain epidermal cells of vertebrates,
and form covalently cross-linked meshworks that persist even after the cell
dies. Hair, fingernails, and feathers are among the structures composed
primarily of keratins.
The Cytoplasm Is Crowded, Highly Ordered, and Dynamic
The picture that emerges from this brief survey is of a eukaryotic cell with
a cytoplasm crisscrossed by a meshwork of structural fibers, throughout which
extends a complex system of membrane-bounded compartments (see Fig. 2-8). Both
the filaments and the organelles are dynamic: the filaments disassemble and
reassemble elsewhere; membranous vesicles bud from one organelle, move to and
join another. l~ansport vesicles, mitochondria, chloroplasts, and other
organelles move through the cytoplasm along protein filaments, drawn by kinesin,
cytoplasmic dynein, myosin, and perhaps other similar proteins. Exocytosis and
endocytosis provide paths between the cell interior and the surrounding medium,
allowing for the secretion of proteins and other components produced within the
cell and the uptake of extracellular components. The intracellular membrane
systems segregate specific metabolic processes, and provide surfaces on which
certain enzyme-catalyzed reactions occur.
Although complex, this organization of the cytoplasm is far from random. The
motion and positioning of organelles and cytoskeletal elements are under tight
regulation, and at certain stages in a eukaryotic cell's life, dramatic, finely
orchestrated reorganizations occur, such as spindle formation, chromatid
migration to the poles, and nuclear envelope disintegration and re-formation
during mitosis. The interactions between the cytoskeleton and organelles are
noncovalent, reversible, and subject to regulation in response to various
intracellular and extracellular signals. Cytoskeletal rearrangements are
modulated by Ca2+ and by a variety of proteins.
Organelles Can Be Isolated by Centrifugation
A major advance in the biochemical study of cells was the development of
methods for separating organelles from the cytosol and from each other. In a
typical cellular fractionation, cells or tissues are disrupted by gentle
homogenization in a medium containing sucrose (about 0.2 M). This
treatment ruptures the plasma membrane but leaves most of the organelles intact.
(The sucrose creates a medium with an osmotic pressure similar to that within
organelles; this prevents diffusion of water into the organelles, which would
cause them to swell, burst, and spill their contents.)
Organelles such as nuclei, mitochondria, and lysosomes differ in size and
therefore sediment at different rates during centrifugation. They also differ in
specific gravity, and they "float" at different levels in a density gradient
(Fig. 2-24). Differential centrifugation results in a rough fractionation of the
cytoplasmic contents, which may be further purified by isopycnic centrifugation.
In this procedure, organelles of different buoyant densities (the result of
different ratios of lipid and protein in each type of organelle) are separated
on a density gradient. By carefully removing material from each region of the
gradient and observing it with a microscope, the biochemist can establish the
position of each organelle and obtain purified organelles for further study. In
this way it was established, for example, that lysosomes contain degradative
enzymes, mitochondria contain oxidative enzymes, and chloroplasts contain
photosynthetic pigments. The isolation of an organelle enriched in a certain
enzyme is often the first step in the purification of that enzyme.
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