Chapters 6

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
The Cytosol Eukaryotic Cells

• eukaryotic cells part of the cytoplasm
• about 55 of the cells 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

• 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

7
Cytoskeleton

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

8
(No Transcript)
9
1. microfilaments thin filaments made up of a
protein called actin -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
In muscle cells motors within filaments made of
myosin slide along filaments containing actin
Muscle Contraction
In amoeba interaction of actin with
myosin causes cellular contraction and pulls the
cells 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 filaments may be the result of myosin
motors attached to organelles
12

• 2. intermediate filaments more permanent part
  of the cytoskeleton than other filaments
• 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
13
2. intermediate filaments function 1. impart
mechanical strength to the cytoskeleton
specialized for bearing tension (like
microfilaments) 2. support cell shape e.g.
forms the axons of neurons 3. anchor 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 - 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

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

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

17
Microtubule Assembly within the 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
-MT disassembly is a mechanism of certain
chemotherapy drugs
18
Non-membranous Organelles
A. Centrioles short cylinders of tubulin - 9
microtubule triplets -called a 90 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 have a
role in mitosis - spindle and chromosome
alignment
19
B. Cilia Flagella

• cilia projections off of the plasma membrane of
  eukaryotic cells covered with PM BUT NOT
  MEMBRANOUS ORGANELLES
• 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
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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 92 array
• cilia is anchored to a basal body just beneath
  the cell surface

21

• flagella resemble cilia but much larger
• 92 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 92 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

23
Cilia, Flagella and Dynein motors

• dynein walking moves flagella and cilia
• dynein protein has two feet that walk along the
  MT
• 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

24
Membranous Organelles

• completely surrounded by a phospholipid bilayer
  similar to the PM surrounding the cell
• allows for isolation of each individual organelle
  - so that the interior of each organelle does not
  mix with the cytosol
• -known as compartmentalization
• BUT - cellular compartments must talk to each
  other
• therefore the cell requires a well-coordinated
  transport system in order for the organelles to
  communicate and function together
• -vesicular transport
• -active process requires ATP

25
Membranous Organelles

• major functions of the organelles
• 1. protein synthesis ER and Golgi
• 2. energy production mitochondria
• 3. waste management lysosomes and peroxisomes

26
Membranous Organelles

• the organelles of a eukaryotic cell are not
  constructed de novo
• they require information in the organelle itself
• when a cell divides it must duplicate its
  organelles also
• in general the cell enlargens existing
  organelles by incorporating new phospholipids and
  proteins into them
• the bigger organelle then divides when the
  daughter cell divides during cytokinesis

27
The Endomembrane System A Review

• endomembrane system is a complex and dynamic
  player in the cells compartmental organization
• divides the cell into compartments
• includes the
• Nucleus
• Endoplasmic Reticulum
• Golgi apparatus
• lysosomes, endosomes
• vacuoles and vesicles

28
The Endomembrane System A Review

• proteins travelling through the ER and Golgi are
  destined for
• 1. Secretion outside the cell
• 2. Plasma membrane
• 3. Lysosome

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• Endoplasmic reticulum (ER) series of
  membrane-bound, flattened sacs in communication
  with the nucleus and the PM
• -each sac or layer cisternae
• -inside or each sac lumen (10 of total cell
  volume)
• -distinct regions of the ER are functionally
  specialized Rough ER vs. Smooth ER

30

• -three functions
• 1. synthesis phospholipids, lipids and proteins
• proteins
• phospholipids lipids
• 2. storage intracellular calcium
• 3. transport site of transport vesicle
  production

31

1. Endoplasmic reticulum (ER)

-two types Rough ER - outside studded with
ribosomes -continuous with the nuclear membrane
-protein synthesis, phospholipid synthesis -also
the initial site of processing and sorting of
proteins
32

1. Endoplasmic reticulum (ER)

-the import of proteins into the RER is a
co-translational process -import of proteins
into an organelle translocation -proteins are
imported as they are being translated by
ribosomes -in contrast to the import of
proteins into other organelles (e.g.
chloroplasts, mitochondria, peroxisomes) and the
nucleus post-translational process
33
Co-translational Protein Synthesis

• two kinds of proteins enter the ER
• ER proteins transmembrane proteins that stay
  stuck in the ER membrane PLUS ER lumen proteins
  that remain in the ER
• 2. proteins destined for the Golgi, PM or
  lysosome or secretion

34
Co-translational Protein Synthesis

• transport from the ribosome across the ER
  membrane requires the presence of an ER signal
  sequence (red in the figure)
• 16-30 amino acids at the beginning of the peptide
  sequence (N-terminal)

35
Co-translational Protein Synthesis

• a complex of proteins will bind this signal in
  the cytoplasm signal recognition particle/SRP
• the ER membrane has receptor for the SRP and
  ribosome SRP receptor (yellow protein in
  figure)
• the ribosome is docked next to a hole in the
  ER membrane (blue protein in figure) translocon
  
• translocon recognizes the signal sequence and
  binds it ? guides the rest of the translating
  polypeptide into the ER lumen
• once the polypeptide is fed into the ER lumen a
  peptidase (located in the SRP receptor complex)
  cleaves the signal sequence off

36
Translocation

• try this animation it might be a bit
  complicated but give it a try anyway
• http//www.rockefeller.edu/pubinfo/proteintarget.h
  tml
• heres a figure from a molecular biology text
  that summarizes the process

37

• once the polypeptide is fed into the ER lumen a
  peptidase cleaves the signal sequence off
  PRODUCES A SOLUBLE PROTEIN
• localizes to the ER lumen
• the presence of another sequence of amino acids
  within the polypeptide stop-transfer sequence
  the translocator stops translocating and
  transfers the polypeptide into the ER membrane
  PRODUCES A TRANSMEMBRANE PROTEIN

38
Modifications in the RER

• 1. folding of the peptide chain
• actually a spontaneous process due to the side
  chains on the amino acids
• only properly folded proteins get transported to
  the Golgi for additional processing and transport
• many proteins located in the ER which supervise
  this folding
• 2. formation of disulfide bonds
• help stabilize the tertiary and quaternary
  structure of proteins

39
Modifications in the RER

• 3. breaking of specific peptide bonds
  proteolytic cleavage or proteolysis
• 4. assembly into multimeric proteins (more than
  one chain)

for an animation go to http//sumanasinc.com/webc
ontent/animations/content/proteinsecretion_mb.html
40
Modifications in the RER

• 5. addition and processing of carbohydrates
  glycosylation
• N-linked glycosylation attachment of 14 sugar
  residues as a group to an asparagine amino acid
  within the protein
• the sugar is actually built and then transferred
  as one unit to the nearby translating protein by
  a transferase protein
• needs to be trimmed down in order to allow
  protein folding

most proteins made in the ER undergo N-linked
glycosylation
41

1. Endoplasmic reticulum (ER)

• Smooth ER extends from the RER
• free of ribosomes
• main function is transport vesicle synthesis
  area where this happens can be called
  transitional ER

42

• but other cell types have SER with enzymes
  embedded in it for additional functions
• lipid and steroid biosynthesis for membranes
• 2. detoxification of toxins and drugs
• 3. cleaves glucose so it can be released into the
  bloodstream
• 4. uptake and storage of calcium

43

• 2. Ribosomes can be considered a nonmembranous
  organelle
• made in the nucleolus
• 2 protein subunits in combination with rRNA
• -large subunit 28S rRNA, 5.8S rRNA, 5 rRNA
  50 proteins
• -small subunit 18S rRNA 33 proteins
• proteins are translating in the cytoplasm and
  imported into the nucleus
• rRNA is transcribed in the nucleolus
• ribosomes found in association with the ER
  where the peptide strand is fed into from the
  ribosome
• also float freely within the cytoplasm as groups
  polyribosomes

44
3. Golgi Apparatus stacks of membranes called
cisternae (cisterna, singular) -the first sac in
the stack cis-face (faces the ER) -the last
sac in the stack trans-face -the ones in the
middle medial cisterna or cisternae
Named after Camillo Golgi in 1897
45
3. Golgi Apparatus -associated with the cis and
trans faces are additional networks of
interconnected cisternal structures -called the
cis Golgi network (CGN) and trans Golgi network
(TGN) -the TGN has a critical role in protein
sorting
46
3. Golgi Apparatus

• site of final protein modification and packaging
  of the finished protein
• functions
• 1. protein modification
• A. glycosylation - creation of glycoproteins and
  proteoglycans
• B. site for phosphate addition to proteins
  phosphorylation
• C. protein trimming
• 2. production of sugars
• Golgi makes many kinds of polysaccharides
• 3. formation of the lysosome
• 4. packaging of proteins and transport to their
  final destination
• TGN acts as a sorting station for transport
  vesicles

47
Modifications in the Golgi

• glycosylation produces a glycoprotein or a
  proteoglycan
• most plasma membrane and secreted proteins have
  one or more carbohydrate chains
• sugars help target proteins to their correct
  location are important in cell-cell and
  cell-matrix interactions
• two kinds N-linked and O linked
• O-linked sugars are added one at a time in the
  Golgi to the amino acids serine, threonine or
  lysine (usually one to four saccharide subunits
  total)
• N-linked sugars are added as a group (about 14
  sugars!) in the ER

48

• glycosylation
• glycosylation starts in the ER
• N-linked glycosylation addition of N-linked
  oligosaccharides
• many of these N-linked sugar residues are trimmed
  off within the ER
• important for folding of the protein
• glycosylation continues in the cisternae of the
  Golgi
• addition of O-linked oligosaccharides to proteins
• PLUS modification of the N-linked
  oligosaccharides - either addition or removal of
  sugar residues

49
Why Glycosylation?

• the vast abundance of glycoproteins suggests that
  glycosylation has an important function
• N-linked is found in all eukaryotes including
  single-celled yeasts
• a type of N-linked can even be found in archaea
  in their cell walls
• WHY GLYCOSYLATION?
• N-linked in the ER is important for proper
  protein folding
• N-linked also limits the flexibility of the
  protein
• the sugar residues can prevent the binding of
  pathogens
• sugar residues also function as signaling
  chemicals
• sugar residues function in cell interactions

50
Why Glycosylation?

• O-linked glycosylation
• O-linked are added one at a time in the Golgi to
  the amino acids serine, threonine or lysine (one
  to four saccharide subunits total)
• added on by enzymes called glycosyltransferases
• human A, B and O antigens are sugars added onto
  proteins and lipids in the plasma membrane of the
  RBC
• everyone has the glycosyltransferase needed to
  produce the O antigen
• those with blood type A have an additional Golgi
  glycosyltransferase enzyme which modifies the O
  antigen to make the A antigen
• a different glycosyltransferase is required to
  make the B antigen
• both glycosyltransferases are required for the
  creation of the AB antigen
• coded for by specific gene alleles on chromosome
  9 (ABO locus)

51
Modifications in the Golgi Protein Trimming

• some PM proteins and most secretory proteins are
  synthesized as larger, inactive pro-proteins that
  will require additional processing to become
  active
• this processing occurs very late in maturation
• processing is catalyzed by protein-specific
  enzymes called proteases
• some proteases are unique to the specific
  secretory protein
• trimming occurs in secretory vesicles that bud
  from the trans-Golgi face
• processing could be at one site (albumin) other
  proteins may require more than one peptide bond
  (insulin)

52
The Golgi Protein transport within the cytoplasm

• protein transport within the cell is tightly
  regulated
• most proteins usually contain tag or signals
  that tell them where to go
• in the Golgi - specific sequences within a
  protein will cause
• 1. retention in the Golgi
• 2. will target it to lysosomes
• 3. send it to the PM for fusion
• 4. send it to the PM for secretion
• a lack of a signal means you will automatically
  be secreted constitutive secretion

53
SO - WHERE DO PROTEINS GO AFTER THE
GOLGI??? -proteins budding off the Golgi have
three targets

• targets
• secretory vesicles for exocytosis
• membrane vesicles for incorporation into PM
• transport vesicles for the lysosome

54
WHAT IF YOU ARENT ONE OF THESE PROTEINS??

• ER proteins stay in the ER
• never traffic to the Golgi
• these ER proteins will have a retention signal
• Ribosomal proteins
• translation of ribosomal proteins are done in the
  cytoplasm by polyribosomes
• assembled into the large and small protein
  subunits in the cytoplasm
• imported into the nucleus
• rRNAs are transcribed in the nucleolus - no
  translation!!!
• protein subunits and rRNAs are assembled in the
  nucleolus to form the small and large ribosomal
  subunits exported from nucleus
• mitochondrial proteins
• the mitochondria has its own DNA, transcribes its
  own mRNA and has its own ribosomes for translation

55
4. Lysosomes garbage disposals -dismantle
debris, eat foreign invaders/viruses taken in by
endocytosis or phagocytosis -also destroy worn
cellular parts from the cell itself and recycles
the usable components autophagy -form by
budding off the trans-Golgi network?? -cell
biologists not really sure exactly how the
lysosome forms
56
4. Lysosomes
-contains powerful enzymes to breakdown
substances into their component parts -over 40
kinds of hydrolytic enzymes -these enzymes are
collectively known as acid hydrolases -acidi
c interior - critical for function of these
enzymes -the hydrolytic enzymes of the lysosome
need to be cleaved first in order to become
enzymatically active -done by the acidity of the
lysosomes interior - acidic interior created and
maintained by a hydrogen pump (H ATPase) that
pumps H into the interior - Active
transport -chloride ions that diffuse in
passively through a chloride channel - forms
hydrochloric acid (HCl)
57

• 4. Lysosomes
• several different kinds of lysosomes diverse in
  shape and size
• types
• lysosome form from the budding and fusion of
  vesicles from the TGN
• these vesicles contain lysosomal enzymes
• early endosome forms through receptor-mediated
  endocytosis from the plasma membrane
• late endosome forms by fusion of early
  endosomes with vesicles containing lysosomal
  enzymes
• endolysosome fusion of a late endosome with a
  pre-existing lysosome
• transforms it into a lysosome
• an endolysosome may be considered an immature
  lysosome

58
Diseases at the Organelle Level Tay Sachs and
lysosomes also known a Hexosaminidase A
deficiency -named after Waren Tay and Bernard
Sachs -key identifying mark cherry red spot in
the retina -lack one of the 40 lysosomal enzymes
hexosaminidase -results in the accumulation of
gangliosides (phospholipid) in the cell membrane
of neurons -death of the neuron results ?
failure of nervous system communication
-infantile form of the disease death by 4
yrs -juvenile form death from 5 to 15
yrs -adult onset not fatal progressive loss
of nervous function -most common in Ashkenazi
Jews, French Canadians and Cajun populations in
Lousiana (same mutation as Jews)
59
5. Mitochondria

• -surrounded by a dual phospholipid bilayer
• an outer mitochondrial membrane
• an inner mitochondrial membrane
• a fluid-filled space mitochondrial matrix
  (contains ribosomes!)
• -the inner membrane is folded into
• folds called cristae
• -these increase the membrane surface area for the
  enzymes of Oxidative Phosphorylation

60

• outer membrane - 50 phospholipid 50 protein
• -very permeable - contains pores for the import
  and export of critical materials
• inner membrane - 20 phospholipid 80 protein
• -less permeable vs. the outer membrane
• -folded extensively to form partitions
  cristae
• -contains proteins that work to create an
  electrochemical gradient
• -contains enzymes that use this gradient for the
  synthesis of ATP
• -also contains pumps to move ATP into the
  cytosol

• matrix - lumen of the mitochondria
• -breakdown of glucose into water and CO2 ends
  here (enzymes of the Transition phase Krebs
  Cycle)

61
Cellular Respiration
-glycolysis -transition phase -citric acid
cycle -electron transport chain
http//biology.about.com/gi/dynamic/offsite.htm?si
tehttp//www.sp.uconn.edu/7Eterry/images/anim/ET
S.html http//biology.about.com/gi/dynamic/offsite
.htm?sitehttp//www.biocarta.com/pathfiles/krebPa
thway.asp http//vcell.ndsu.nodak.edu/animations/e
tc/movie.htm
62
6. Peroxisomes found in all cells but abundant
in liver and kidney cells
-only identified in 1954 -may arise from
pre-existing peroxisomes or may bud from the ER
-major function is oxidation (breakdown) of long
chain fatty acids (beta-oxidation) -results in
the conversion of the fatty acid into acetyl coA
? Krebs cycle -in plant cells beta-oxidation
is only done by the peroxisome -in animal cells
the mitochondria can also perform this
reaction -oxidation is done by oxidases enzymes
that use oxygen to oxidize substances -remove
hydrogen atoms from the fatty acid -this reaction
generates hydrogen peroxide (H2O2)
63
6. Peroxisomes found in all cells but abundant
in liver and kidney cells
PROBLEM 1 H2O2 is very corrosive -therefore
peroxisomes also contain an enzyme called
catalase to break this peroxide down into water
and oxygen PROBLEM 2 the electron transport
chain in mitochondria produces superoxide
radicals (O2-) as a normal consequence of
electron leaking (from complex I) -peroxisomes
also contain anti-oxidant enzymes to break down
other dangerous oxidative chemicals made by the
cell during metabolism e.g. SOD breaks down
O2- to make H2O2
-other functions of peroxisomes 1. synthesis
of bile acids 2. breakdown of alcohol by liver
cells
64
Adrenoleukodystrophy and peroxisomes -X linked
disorder in the gene ABCD1 (transporter
protein) -120,000 to 150,000 births -in ALD -
peroxisomes lack an essential enzyme -leads to a
build up of a long-chain saturated fatty acids on
cells of throughout the body -can results in the
loss of the myelin sheath not known
why -lethargy, skin darkens, blood sugar drops,
altered heart rhythm imbalanced electrolytes,
paralysis, death slowed by a certain
triglyceride found in rapeseed oil Lorenzo
Odone Lorenzos Oil (mixture of unsaturated
fatty acids that slows the
development of these saturated FAs)
F-actin and peroxisomes