Chapters 6 & 12

1. Chapters 6 & 12 The Cytoplasm

2. The Typical Cell • typical cell: 1. nucleus • 2. cell membrane • 3. cytoplasm • -cytosol • -cytoskeleton • 4. cytoplasmic organelles • -membranous • -non-membranous

3. Cytoplasm • semi-fluid-like jelly within the cell • division into three subdivisions: cytosol, cytoskeleton & organelles

4. Cytosol ECF • lower K+ • higher Na+ • lower concentration • of dissolved and • suspended proteins • higher concentration • of carbohydrates • smaller reserves of amino • acids • 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) 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

5. 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

6. The Cytoskeleton and Cell motility Vesicle ATP Receptor formotor protein • 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 vesicles to “walk” the vesicle along the cytoskeleton • 2. motor protein, the cytoskeleton and the plasma membrane interact to move the entire cell along the ECM • 3. motor proteins result in the bending of cilia and flagella Microtubuleof cytoskeleton Motor protein(ATP powered) (a) Microtubule Vesicles 0.25 m (b)

7. Cytoskeleton: • three major components • 1. microfilaments • 2. intermediate filaments • 3. microtubules

8. 5 m 10 m 10 m Column of tubulin dimers Keratin proteins Actin subunit Fibrous subunit (keratinscoiled together) 25 nm 812 nm 7 nm Tubulin dimer  

9. 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

10. 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)

11. Examples of Actin/Myosin: Muscle cell 0.5 m Actin In muscle cells – motors within filaments made of myosin “slide” along filaments containing actin = Muscle Contraction filament Myosin filament Myosin head (a) Myosin motors in muscle cell contraction Cortex (outer cytoplasm):gel with actin network 100 m Inner cytoplasm: solwith actin subunits 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) Extendingpseudopodium (b) Amoeboid movement 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 Chloroplast 30 m (c) Cytoplasmic streaming in plant cells

12. 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

13. 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

14. 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 • components of: • mitotic spindle • cilia and flagella • axons of neurons b-tubulin a-tubulin

15. 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 b-tubulin a-tubulin

16. -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

17. 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

18. 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 rolein 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

19. 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

20. 0.1 m Plasma membrane Outer microtubuledoublet B. Cilia & Flagella Dynein proteins Centralmicrotubule • 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 Radialspoke Microtubules Cross-linkingproteins betweenouter doublets (b) Cross section ofmotile cilium Plasmamembrane Basal body 0.1 m 0.5 m (a) Longitudinal sectionof motile cilium Triplet -in certain cells cilia can also function as “antennae” -in these cells there is only one cilium – primary cilium (c) Cross section ofbasal body

21. 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

22. 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 Microtubuledoublets Cross-linking proteinsbetween outer doublets Dynein protein

23. Microtubuledoublets Cilia, Flagella and Dynein “motors” ATP • 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 Dynein protein (a) Effect of unrestrained dynein movement Cross-linking proteinsbetween outer doublets ATP Anchoragein cell (b) Effect of cross-linking proteins 1 3 2 (c) Wavelike motion

24. 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

25. 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

26. 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

27. 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 m

28. 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 Sisterchromatids Centromere 0.5 m • 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

29. 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

30. 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

31. 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

32. 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

33. Mitosis is conventionally divided into five phases • Prophase • Prometaphase • Metaphase • Anaphase • Telophase • Cytokinesis overlaps the latter stages of mitosis

35. 10 m G2 of Interphase Prophase Prometaphase Metaphase Anaphase Telophase and Cytokinesis Centrosomes(with centriole pairs) Chromatin(duplicated) Fragments of nuclearenvelope Nonkinetochoremicrotubules Early mitoticspindle Aster Metaphase plate Cleavagefurrow Nucleolusforming Centromere Plasmamembrane Nuclearenvelope Chromosome, consistingof two sister chromatids Kinetochore Kinetochoremicrotubule Nucleolus Nuclearenvelopeforming Daughterchromosomes Centrosome atone spindle pole Spindle

36. Mitosis 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 spindleforms 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

37. Spindle Formation

38. Outer Plate Microtubules Inner Plate 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 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 Microtubules Prometaphase Fragments of nuclearenvelope Nonkinetochoremicrotubules Kinetochore Kinetochore Kinetochoremicrotubule

39. 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

40. 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

41. 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

42. (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 100 m Cleavage furrow Daughter cells Contractile ring ofmicrofilaments

43. 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 (b) Cell plate formation in a plant cell (TEM) 10 m Cell plate Vesiclesformingcell plate Wall of parent cell 1 m New cell wall Cell plate 5 Telophase Daughter cells

46. Cell wall Origin ofreplication Binary Fission in Bacteria Plasma membrane E. coli cell Bacterial chromosome 1 Chromosomereplicationbegins. Two copies of origin • 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 2 Origin Origin Replicationcontinues. 3 Replicationfinishes. 4 Two daughtercells result.

47. Diatoms andsome yeasts (c) Bacterialchromosome The Evolution of Mitosis (a) Bacteria Chromosomes • mitosis probably evolved from binary fission • certain protists exhibit types of cell division that seem intermediate between binary fission and mitosis Microtubules (b) Dinoflagellates Intact nuclearenvelope Kinetochoremicrotubule Intact nuclearenvelope Kinetochoremicrotubule (d) Most eukaryotes Fragments ofnuclear envelope