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

• 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

The Cytosol – Eukaryotic Cells Cytosol• higher K+• lower Na+• higher concentrationof 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 concentrationof 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

ATPVesicle

(a)

Motor protein(ATP powered)

Microtubuleof cytoskeleton

Receptor formotor protein

0.25 m VesiclesMicrotubule

(b)

• 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• some roles of motor proteins:

• 1. motor proteins interact with microtubules (or microfilaments) and vesiclesto “walk” the vesicle along the cytoskeleton• 2. motor protein, the cytoskeleton andthe plasma membrane interact to move the entire cell along the ECM• 3. motor proteins result in thebending of cilia and flagella

The Cytoskeleton and Cell motility

• three major components1. microfilaments2. intermediate filaments3. microtubules

Cytoskeleton:

Column of tubulin dimers

Tubulin dimer

25 nm

Actin subunit

7 nm

Keratin proteins

812 nm

Fibrous subunit (keratinscoiled together)

10 m 10 m 5 m

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 withinthe cell4. give the cell its shape5. also provide support for cellular extensions called microvilli (small intestines)

Muscle cell

ActinfilamentMyosin

Myosinfilament

head(a) Myosin motors in muscle cell contraction

0.5 m

100 m

Cortex (outer cytoplasm):gel with actin network

Inner cytoplasm: solwith actin subunits

(b) Amoeboid movement

Extendingpseudopodium

30 m(c) Cytoplasmic streaming in plant cells

Chloroplast

In muscle cells – motors within filamentsmade of myosin “slide” along filamentscontaining actin = Muscle Contraction

Examples of Actin/Myosin:

In amoeba – interaction of actin with myosincauses cellular contraction and pulls the cell’s trailing edge (left) forward-can also result in the production of Pseudopodia (for locomotion, feeding)

In plant cells – a layer of cytoplasm cycles around the cell-streaming over a “carpet” of actin filamentsmay be the result of myosin motors attachedto 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 keratinstype II IFs = basic keratinstype III IFs = desmin, vimentintype IV IFs = neurofilamentstype 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 shapee.g. forms the axons of neurons

3. anchors & stabilize organellese.g. anchors the nucleus in place

4. transport materialse.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 & movement3. mitosis - form the spindle (chromosome movement)4. form many of the non-membranous organelles

- cilia, flagella, centrioles

-tubulin

-tubulin

components of:1. mitotic spindle2. cilia and flagella3. 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

• can be found as a single tubea doublet and a triplet

-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 centrioles2. pericentriolar material – made up of factors that mediate microtubule assembly3. “-” end of assembling microtubules (MTs grow out from the centrosome)

-other eukaryotes – there is no MTOC-have other centers for MT assembly

-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

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

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

Non-membranous Organelles

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

cleaninge.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

Microtubules

Plasmamembrane

Basal body

Longitudinal sectionof motile cilium

(a)0.5 m 0.1 m

0.1 m

(b) Cross section ofmotile cilium

Outer microtubuledoubletDynein proteins

CentralmicrotubuleRadialspoke

Cross-linkingproteins betweenouter doublets

Plasma membrane

Triplet

(c) Cross section ofbasal body

-in certain cells cilia can alsofunction as “antennae” -in these cells there is onlyone cilium – primary cilium

• 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”

Dynein protein

Cross-linking proteinsbetween outer doublets

Microtubuledoublets

• 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

• 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 cross-linking 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

Cilia, Flagella and Dynein “motors” Microtubuledoublets

Dynein protein

ATP

(a) Effect of unrestrained dynein movement

Cross-linking proteinsbetween outer doublets ATP

Anchoragein cell

(b) Effect of cross-linking proteins

(c) Wavelike motion

12

3

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

-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

Some terms to know

every eukaryotic species has a characteristic number of chromosomes in each cell nucleus

e.g. humans – n=23e.g. drosophila – n=2e.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 non-

identical 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

20 m

• 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

0.5 mCentromere

Sisterchromatids

• 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 ofsister chromatids during mitosis & meiosis • shared by sister chromatids during mitosis• site where spindle microtubules attach – areaof DNA and protein = kinetochore

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_freeman_dna_coiling.htm

• 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

Eukaryotic Cell Division = Mitosis

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.wisc-online.com/objects/index.asp?objID=AP13604

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.wisc-online.com/objects/index.asp?objID=AP13604

• Mitosis is conventionally divided into five phases

– Prophase– Prometaphase– Metaphase– Anaphase– Telophase

• Cytokinesis overlaps the latter stages of mitosis

G2 of Interphase Prophase PrometaphaseCentrosomes(with centriole pairs)

Chromatin(duplicated)

Nucleolus Nuclearenvelope

Plasmamembrane

Early mitoticspindle

AsterCentromere

Chromosome, consistingof two sister chromatids

Fragments of nuclearenvelope

Nonkinetochoremicrotubules

Kinetochore Kinetochoremicrotubule

Metaphase

Metaphase plate

Anaphase Telophase and Cytokinesis

Spindle Centrosome atone spindle pole

Daughterchromosomes

Cleavagefurrow

Nucleolusforming

Nuclearenvelopeforming

10

m

Mitosis1. Prophase: prior to prophase, the replicated DNA is beginning to

condense into sister chromatids joined at the centromere (duplicated)chromosome1. DNA/chromatin condenses to become visible2. the centrioles (replicated at G2) move apart from each other3. the spindle forms between the centrioles (microtubules)-the centrioles are not essential for spindle formation; plant cells do nothave centrioles-spindle MT assembly results from the polymerization of tubulin subunuts-other MTs of the cytoskeleton disassemble to provide more tubulin to the spindle4. the centrioles migrate to opposite poles of the cell5. the nucleoli disappear

http://www.loci.wisc.edu/outreach/bioclips/CDBio.html

Spindle – structure that includes thetwo centrioles, two asters and the spindlemicrotubules than span the cell

Aster – a radial array of short MTs extending from thecentrioles

Spindle Formation

2. Prometaphase: used to be known as “late prophase”-the DNA has condensed into sister chromatids joined at the centromere (duplicated)chromosome1. the nuclear envelope fragments – allows growth of spindle

into regionwhere chromosomes are located2. the DNA becomes even more condensed3. some chromosomes attach to spindle via kinetochore =

kinetochore microtubules4. non-kinetochore microtubules begin to form and grow towards

opposite polePrometaphase

Fragments of nuclearenvelope

Nonkinetochoremicrotubules

Kinetochore Kinetochoremicrotubule

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

Inner Plate

Outer Plate

Microtubules

Kinetochore

Chromatid

Microtubules

3. Metaphase: centrioles are at opposite ends of the cell and the spindleis complete1. the chromosomes move and line up along a central zone= metaphase plate-the tug of war at pro-metaphase eventually positions the chromosomesmidway alone the length of the cell2. non-kinetochore MTs interact with the opposite pole, the aster MTs make contact with the plasma membrane – the spindle is now complete

10 m

Metaphase3

4. Anaphase: shortest of the mitotic phases1. the chromatid pairs separate into daughter chromosomes2. one chromatid/chromosome moves toward one centriole of the cell, the other the opposite-pulled apart by the action of the spindle – the kinetochore MTsbegin to shorten3. 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 separatebecause of enzymatic activity-an enzyme called separase cleavesa protein known as cohesin (proteinin the centromere that holds thesister chromatids togeter)-separates the sister chromatids

4. Telophase: reverse of Prophase1. 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 cell2. the nucleoli reappear3. the spindle disappears as the MTs depolymerize4. daughter chromosomes uncoil

** Cytokinesis starts during late anaphase and is well underway during telophase

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

(a) Cleavage of an animal cell (SEM)

Cleavage furrow

Contractile ring ofmicrofilaments

Daughter cells

100 m

(b) Cell plate formation in a plant cell (TEM)

Vesiclesformingcell plate

Wall of parent cell

Cell plate New cell wall

Daughter cells

1 m

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

10 m

Telophase5

Cell plate

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

1

Origin ofreplication

E. coli cell

Two copies of origin

Cell wallPlasma membrane

Bacterial chromosome

Origin Origin

Chromosomereplicationbegins.

Replicationcontinues.

Replicationfinishes.

Two daughtercells result.

2

3

4

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

(b) Dinoflagellates

(d) Most eukaryotes

Intact nuclearenvelope

Chromosomes

Microtubules

Intact nuclearenvelope

Kinetochoremicrotubule

Kinetochoremicrotubule

Fragments ofnuclear envelope

Bacterialchromosome

(c)Diatoms andsome yeasts