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Chapters 6 & 12
The Cytoplasm
The Typical Cell
• typical cell: 1. nucleus
2. cell membrane
3. cytoplasm
-cytosol
-cytoskeleton
4. cytoplasmic organelles
-membranous
-non-membranous
Cytoplasm
•
•
semi-fluid-like jelly within the cell
division into three subdivisions: cytosol, cytoskeleton &
organelles
The Cytosol – Eukaryotic Cells
• eukaryotic cells – part of the cytoplasm
– about 55% of the cell’s volume
– about 70-90% water PLUS
•
•
•
•
•
ions
dissolved nutrients – e.g. glucose
soluble and insoluble proteins
waste products
macromolecules and their components - amino acids, fatty
acids
• ATP
• unique composition with respect to extracellular
fluids
Cytosol
•higher K+
•lower Na+
•higher concentration
of dissolved and
suspended proteins
(enzymes, organelles)
•lower concentration
of carbohydrates
(due to catabolism)
•larger reserves of amino
acids (anabolism)
ECF
•lower K+
•higher Na+
•lower concentration
of dissolved and
suspended proteins
•higher concentration
of carbohydrates
•smaller reserves of amino
acids
Cytoskeleton:
•internal framework of the cell
•gives the cytoplasm flexibility and strength
•provides the cell with mechanical support
•gives the cell its shape
•can be rapidly disassembled in one area of
the cell and reassembled in another
•anchorage points for organelles and cytoplasmic
enzymes
•also plays a role in cell migration and movement
by the cell
The Cytoskeleton and Cell motility
• motility = changes in cell location and the limited movements in
parts of the cell
• the cytoskeleton is involved in many types of motility
• requires the interaction of the cytoskeleton with motor proteins
Vesicle
• some roles of motor proteins:
ATP
• 1. motor proteins interact with
microtubules (or microfilaments) and vesicles
to “walk” the vesicle along the cytoskeleton
(a)
• 2. motor protein, the cytoskeleton and
Microtubule
the plasma membrane interact to
move the entire cell along the ECM
• 3. motor proteins result in the
bending of cilia and flagella
(b)
Receptor for
motor protein
Motor protein
(ATP powered)
Vesicles
Microtubule
of cytoskeleton
0.25 m
Cytoskeleton:
•three major components
1. microfilaments
2. intermediate filaments
3. microtubules
10 m
10 m
5 m
Column of tubulin dimers
Keratin proteins
Fibrous subunit (keratins
coiled together)
Actin subunit
25 nm
7 nm


Tubulin dimer
812 nm
1. microfilaments = thin filaments made up of a protein called actin
-solid rods of about 7nm
-twisted double chain of actin subunits
-forms a dense network immediately under the PM (called the cortex)
-also found scattered throughout the cytoplasm
1. microfilaments =
-function: 1. anchor integral proteins and attaches them to the cytoplasm
2. interaction with myosin = interacts with larger microfilaments made up of myosin
- results in active movements within a cell (e.g. muscle cell contraction)
3. provide much of the mechanical strength of the cell – resists pulling forces within
the cell
4. give the cell its shape
5. also provide support for cellular extensions called microvilli (small intestines)
Examples of Actin/Myosin:
Muscle cell
0.5 m
Actin
filament
In muscle cells – motors within filaments
made of myosin “slide” along filaments
containing actin = Muscle Contraction
Myosin
filament
Myosin
head
(a) Myosin motors in muscle cell contraction
Cortex (outer cytoplasm):
gel with actin network
100 m
Inner cytoplasm: sol
with actin subunits
Extending
pseudopodium
In amoeba – interaction of actin with myosin
causes cellular contraction and pulls the
cell’s trailing edge (left) forward
-can also result in the production of
Pseudopodia (for locomotion, feeding)
(b) Amoeboid movement
Chloroplast
(c) Cytoplasmic streaming in plant cells
30 m
In plant cells – a layer of cytoplasm cycles
around the cell
-streaming over a “carpet” of actin filaments
may be the result of myosin motors attached
to organelles
2. intermediate filaments = more permanent part of the cytoskeleton than other
filaments
- range from 8 to 12 nm in diameter
-five types of IF filaments – type I to type V
-made up of proteins such as vimentin, desmin, or keratin
-each cell type has a unique complement of IFs in their cytoskeleton
- all cells have lamin IFs – but these are found in the nucleus
-some cells also have specific IFs
-
e.g neurons also posses IFs made of neurofilaments
type I IFs = acidic keratins
type II IFs = basic keratins
type III IFs = desmin, vimentin
type IV IFs = neurofilaments
type V IFs = nuclear lamins
kidney cell - vimentin
2. intermediate filaments =
function: 1. impart strength to the cytoskeleton – specialized for bearing tension (like
microfilaments)
2. support cell shape
e.g. forms the axons of neurons
3. anchors & stabilize organelles
e.g. anchors the nucleus in place
4. transport materials
e.g. movement of neurotrasmitters
into the axon terminals
3. microtubules = hollow rods or “straws” of 25 nm in diameter
- made of repeating units of proteins called tubulin
- function: 1. cell shape & strength
2. organelles: anchorage & movement
3. mitosis - form the spindle (chromosome movement)
4. form many of the non-membranous organelles
- cilia, flagella, centrioles
-tubulin
-tubulin
components of:
1. mitotic spindle
2. cilia and flagella
3. axons of neurons
3. microtubules -the basic microtubule is a hollow cylinder = 13 rows of tubulin called
protofilaments
-tubulin is a dimer – two slightly different protein subunits
- called alpha and beta-tubulin
-alternate down the protofilament row
-tubulin
-tubulin
-animal cells – microtubule assembly occurs in the MTOC (microtubule organizing
center or centrosome)
-area of protein located near the nucleus
-within the MTOC/centrosome :
1. a pair of modified MTs called centrioles
2. pericentriolar material – made up of factors that mediate microtubule
assembly
3. “-” end of assembling microtubules (MTs grow out from the centrosome)
-other eukaryotes – there is no MTOC
-have other centers for MT assembly
•can be found as a single tube
a doublet and a triplet
Microtubule Assemby:
-done within the MTOC or a region of the cell that functions as an MTOC
-MTs are easy to assemble and disassemble – by adding or removing tubulin dimers
-one end accumulates or releases tubulin dimers much faster than the other end called the
plus end
-the tubulin subunits bind and hydrolyze GTP – determines how they polymerize into the MT
-tubulin subunits bound to GTP or GDP-Pi
are very stable – can’t add onto them
-act as “caps” to prevent the
disassembly of the microtubule
-hydrolysis of GTP GDP + Pi and the
loss of the Pi group allows for the
addition of another tubulin subunit
-MUST add another tubulin onto this
GDP-bound tubulin end or the MT will
disassemble
-mechanism is the target of
chemotherapy drugs
http://www.nature.com/nrc/journal/v4/n4/fig_tab/nrc1317_F4.html
Non-membranous Organelles
A. Centrioles: short cylinders of tubulin
- 9 microtubule triplets
-called a 9+0 array (9 peripheral triplets, 0 in the center)
-grouped together as pairs – arranged perpendicular to one another
-make up part of the centrosome or MTOC
-role in MT assembly??
-also has role in mitosis - spindle and chromosome alignment
-found near the Golgi apparatus during interphase
-duplicate just prior to the onset of mitosis
-migrate to opposite ends of the replicating cell
-spindle of MTs grows in between
B. Cilia & Flagella
• cilia = projections off of the plasma membrane of eukaryotic cells – covered with PM BUT
NOT MEMBRANOUS ORGANELLES
• about 0.25um in diameter and only 20um long
• beat rhythmically to transport material – power & recovery strokes
• found in linings of several major organs covered with mucus where they function in
cleaning
e.g. trachea, lungs
Trachea
B. Cilia & Flagella
• cytoskeletal framework of a cilia or flagella = axoneme (built of microtubules)
• contain 9 groups of microtubule doublets surrounding a central pair= called a 9+2 array
• cilia is anchored to a basal body just beneath the cell surface
0.1 m
Outer microtubule
doublet
Dynein proteins
Central
microtubule
Radial
spoke
Microtubules
Plasma
membrane
Cross-linking
proteins between
outer doublets
(b) Cross section of
motile cilium
Basal body
-in certain cells cilia can also
function as “antennae”
-in these cells there is only
one cilium – primary cilium
0.5 m
(a) Longitudinal section
of motile cilium
0.1 m
Triplet
(c) Cross section of
basal body
Plasma membrane
•flagella = resemble cilia but much larger
• 9+2 array
• found singly per cell
• functions to move a cell through the ECF
-DO NOT HAVE THE SAME STRUCTURE AS BACTERIAL FLAGELLA
Cilia, Flagella and Dynein “motors”
• in flagella and motile cilia – flexible cross-linked
proteins are found evenly spaced along the
length
– blue in the figure
• these proteins connect the outer doublets to
each other and to the two central MTs of a 9+2
array
• each outer doublet also has pairs of proteins
along its length
– these stick out and reach toward its neighboring
doublet
– called dynein motors
– responsible for the bending of the microtubules of
cilia and flagella when they beat
Microtubule
doublets
Cross-linking proteins
between outer doublets
Dynein protein
Cilia, Flagella and Dynein “motors”
•
dynein “walking” moves flagella and cilia
− dynein protein has two “feet” that walk along the MT
− ATP provides the energy
− dyneins alternately grab, move, and release the outer
microtubules
− BUT: without any cross-linking between adjacent MTs
- one doublet would slide along the other
− elongate the cilia or flagella rather than bend it
− so to bend the MT  must have proteins crosslinking between the MT doublets (blue lines in
figure)
– protein cross-links limit sliding
– forces exerted by dynein walking causes doublets to
curve = bending the cilium or flagellum
– bending starts at the base and moves to the tip
– wavelike motion results depending on which MT
doublets bend
Microtubule
doublets
ATP
Dynein protein
(a) Effect of unrestrained dynein movement
Cross-linking proteins
between outer doublets
ATP
Anchorage
in cell
(b) Effect of cross-linking proteins
1
3
2
(c) Wavelike motion
Centrioles, Spindles & Cell Division
• the presence of centrioles in some eukaryotic
cells is indicative of cells capable of division – or
Mitosis
• in unicellular organisms, division of one cell
reproduces the entire organism
– through a process called fission
• multicellular organisms depend on cell division
for
– 1. development from a fertilized cell
– 2. growth
– 3. repair
• mitosis is an integral part of the cell cycle, the life
of a cell from formation to its own division
Some terms to know
-parent cell - cell about to undergo
division
-daughter cell – cell that results from
either mitosis or meiosis
-somatic cell = any cell within the body
other than an egg or sperm
somatic cell has two complete sets of
chromosomes
-one set is called the haploid
number of chromosomes (n)
-therefore the cell is said to be
diploid (2n)
e.g. humand n = 23 (2n = 46)
-germ cell or gamete = sex cell
-gamete has only one set of
chromosomes and is haploid
every eukaryotic species has a characteristic
number of chromosomes in each cell nucleus
e.g. humans – n=23
e.g. drosophila – n=2
e.g. dog – n=39
Most cell division results in genetically identical
daughter cells
• most cell division results in daughter cells with identical genetic
information (i.e. amount and type of DNA)
– the exception is meiosis – a modified division process that produces nonidentical daughter cells called sperm and egg
– these cells have half the amount of genetic information
• the genetic information has to be duplicated and distributed
amongst the two daughter cells
• once the DNA is duplicated and distributed then the cell can
divide
• SO: cell division is not just the pinching of the parent cell into two
daughter cells
Cellular Organization of the Genetic
Material
• all the DNA in a cell constitutes the cell’s genome
• REMINDER: eukaryotic chromosomes consist of chromatin, a
complex of DNA and protein that condenses during cell division
• in most cells - DNA molecules in a cell are
condensed and packaged into chromosomes
• prokaryotics have a single chromosome called a
genophore
• eukaryotic cells posses number of
chromosomes
20 
• when not dividing – eukaryotic DNA is in its loosest formation – chromatin
– allows access to the machinery for DNA replication and transcription
• in preparation for cell division, - DNA is replicated and condenses into
chromosomes
• chromosome = organized structure of DNA and protein
– chroma = color
– soma = body
• the building material of a chromosome is chromatin
• each duplicated chromosome is made of two sister chromatids = joined
copies of the original chromosome
– these chromatids will separate during cell division and be partitioned into each daughter cell
• chromatids are joined by a structure called a centromere
Centromere
• condensed regions within the chromosome
• responsible for the accurate segregation of
sister chromatids during mitosis & meiosis
• shared by sister chromatids during mitosis
• site where spindle microtubules attach – area
of DNA and protein = kinetochore
Sister
chromatids
Centromere
0.5 m
Chromosome and Chromosomes:
Confusion!!!
• prior to cell division – the duplicated
chromatin condenses into its most
dense form = chromosome
– two sister chromatids joined by a
centromere
– typically called a duplicated chromosome
• during cell division - the two sister
chromatids separate
• once separated - the chromatids are
still called chromosomes
DNA condensation animation -
http://www.biostudio.com/demo_fr
eeman_dna_coiling.htm
Eukaryotic Cell Division = Mitosis
• eukaryotic cell division consists of
– Mitosis - the division of the genetic material in the nucleus
– Cytokinesis - the division of the cytoplasm
• mitosis described by the German anatomist Walther
Flemming in 1882
– thought the cell was simply growing larger between each
period of cell division
• now known that mitosis is a part of the life cycle of a cell
• called the Cell Cycle
– internal “clock” that defines the periods of DNA synthesis and
replication
Phases of the Cell Cycle
• consists of two phases
– Mitotic (M) phase = mitosis and
cytokinesis)
– Interphase = cell growth and copying
of chromosomes in preparation for
cell division
• Interphase - about 90% of the cell
cycle
–
–
–
–
–
can be divided into sub-phases
G1 phase -“first gap”
S phase “synthesis”
G2 phase - “second gap”
progression from one phase to
another is called a checkpoint
• major checkpoints are : G1/S & G2/M
http://www.wisconline.com/objects/in
dex.asp?objID=AP136
04
Phases of the Cell Cycle
– G1 phase - time in phase depends on species
•
•
•
•
•
normal cell functions
growth in size
duplication of organelles
mRNA and protein synthesis in preparation for S phase
critical phase in which cell commits to division or leaves
the cell cycle to enter into a dormancy phase (G0)
– S phase - 6 to 8 hours
•
•
•
synthesis of histone proteins & DNA replication
chromatin assembly
correction of DNA damage
– G2 phase – 2 to 5 hours
•
may not be necessary in all cells
–
•
•
•
•
e.g. cancer cells
rapid cell growth – may function to simply control cell
size
protein synthesis in preparation for M phase
duplication of the centrioles/centrosomes
G2/M checkpoint verifies correction of DNA damage
http://www.wisconline.com/objects/in
dex.asp?objID=AP136
04
• Mitosis is conventionally
divided into five phases
–
–
–
–
–
Prophase
Prometaphase
Metaphase
Anaphase
Telophase
• Cytokinesis overlaps the
latter stages of mitosis
10 m
G2 of Interphase
Centrosomes
(with centriole pairs)
Nucleolus
Chromatin
(duplicated)
Nuclear
envelope
Plasma
membrane
Prophase
Early mitotic
spindle
Aster
Centromere
Chromosome, consisting
of two sister chromatids
Prometaphase
Fragments
of nuclear
envelope
Kinetochore
Metaphase
Nonkinetochore
microtubules
Kinetochore
microtubule
Anaphase
Metaphase
plate
Spindle
Centrosome at
one spindle pole
Telophase and Cytokinesis
Cleavage
furrow
Daughter
chromosomes
Nuclear
envelope
forming
Nucleolus
forming
Mitosis
1.
http://www.loci.wisc.edu/outreach/bioclips/CDBio.html
Prophase: prior to prophase, the replicated DNA is beginning to condense into
sister chromatids joined at the centromere  (duplicated)chromosome
1. DNA/chromatin condenses to become visible
2. the centrioles (replicated at G2) move apart from each other
3. the spindle forms between the centrioles (microtubules)
-the centrioles are not essential for spindle formation; plant cells do not
have centrioles
-spindle MT assembly results from the polymerization of tubulin subunuts
-other MTs of the cytoskeleton disassemble to provide more tubulin to the spindle
4. the centrioles migrate to opposite poles of the cell
5. the nucleoli disappear
Spindle – structure that includes the
two centrioles, two asters and the spindle
microtubules than span the cell
Aster – a radial array of short MTs extending from the
centrioles
Spindle Formation
2. Prometaphase: used to be known as “late prophase”
-the DNA has condensed into sister chromatids joined at the centromere
 (duplicated)chromosome
1. the nuclear envelope fragments – allows growth of spindle into region
where chromosomes are located
2. the DNA becomes even more condensed
3. some chromosomes attach to spindle via kinetochore = kinetochore
microtubules
4. non-kinetochore microtubules begin to form and grow towards opposite
pole
Prometaphase
Fragments
of nuclear
envelope
Nonkinetochore
microtubules
Kinetochore – a structure of DNA (CEN DNA) and proteins located in
the centromere
-for the attachment of the spindle to the chromosome
-one MT attaches to one kinetochore on one chromatid
-a 2nd MT attaches to the kinetochore on the other chromatid
-attachment of these MTs results in movement toward the poles
-a “tug of war” results – chromosomes move back and forth
-mutations in the CEN DNA can abolish the ability to segregate
Chromatid
Outer Plate
Microtubules
Microtubules
Kinetochore
Kinetochore
microtubule
Kinetochore
Inner Plate
3.
Metaphase: centrioles are at opposite ends of the cell and the spindle
is complete
1. the chromosomes move and line up along a central zone= metaphase plate
-the tug of war at pro-metaphase eventually positions the chromosomes
midway alone the length of the cell
2. non-kinetochore MTs interact with the opposite pole, the aster MTs
make contact with the plasma membrane – the spindle is now complete
10 m
3 Metaphase
4. Anaphase: shortest of the mitotic phases
1. the chromatid pairs separate into daughter chromosomes
2. one chromatid/chromosome moves toward one centriole of the cell,
the other the opposite
-pulled apart by the action of the spindle – the kinetochore MTs
begin to shorten
3. non-kinetochore MTs grow – this elongates the cell
** At the end of this phase – each end of the cell has equivalent numbers
of chromosomes – same number as the parent cell
**the sister chromatids separate
because of enzymatic activity
-an enzyme called separase cleaves
a protein known as cohesin (protein
in the centromere that holds the
sister chromatids togeter)
-separates the sister chromatids
4. Telophase: reverse of Prophase
1. nuclear envelope reforms – two daughter nuclei result
-part of the new nuclear membrane is recycled from the old fragments,
other parts are made new by the cell
2. the nucleoli reappear
3. the spindle disappears as the MTs depolymerize
4. daughter chromosomes uncoil
** Cytokinesis starts during late anaphase and is well underway during
telophase
(a) Cleavage of an animal cell (SEM)
Cytokinesis: division of cytoplasm
-separates the parent into two daughter cells
-differs in animal cells and plant cells
Animal cell Cytokinesis: results from
cleavage -pinches into two daughters
-actin filaments assemble to form a
contractile ring along the equator of the
cell
-actin interacts with myosin proteins –
causes the ring to contract
-forms a “cleavage furrow” - slight
indentation around the circumference of
the cell
-cell divides by a “purse string”
mechanism
Cleavage furrow
Contractile ring of
microfilaments
100 m
Daughter cells
Plant cell Cytokinesis: No cleavage furrow possible
-vesicles bud from the Golgi apparatus and migrate to the middle of the cell
-vesicles coalesce to produce a cell plate
-other vesicles fuse to the plate bringing in new building materials
-cell plate grows and eventually splits the cell into two daughter cells
Cell plate
10 m
(b) Cell plate formation in a plant cell (TEM)
Vesicles
forming
cell plate
5 Telophase
Wall of parent cell
Cell plate
1 m
New cell wall
Daughter cells
Binary Fission in Bacteria
• bacteria and archaea
reproduce by binary fission
– the chromosome replicates
and the two daughter
chromosomes actively move
apart
– the plasma membrane
pinches inward, dividing the
cell into two
Origin of
replication
E. coli cell
1 Chromosome
replication
begins.
2 Replication
continues.
3 Replication
finishes.
4 Two daughter
cells result.
Cell wall
Plasma membrane
Bacterial chromosome
Two copies
of origin
Origin
Origin
The Evolution of Mitosis
• mitosis probably
evolved from binary
fission
• certain protists
exhibit types of cell
division that seem
intermediate
between binary
fission and mitosis
(a) Bacteria
Bacterial
chromosome
Chromosomes
(b) Dinoflagellates
Microtubules
Intact nuclear
envelope
Kinetochore
microtubule
(c)Diatoms and
some yeasts
Intact nuclear
envelope
Kinetochore
microtubule
(d) Most eukaryotes
Fragments of
nuclear envelope