Chapters 6

The Cytoplasm & Organelles

The Typical CellThe Typical Cell• typical cell: 1. nucleus

2. cell membrane 3. cytoplasm -cytosol

-cytoskeleton 4. cytoplasmic organelles

-membranous-non-membranous

CytoplasmCytoplasm• 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 CellsThe 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 motilityThe 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-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

Actinfilament

Myosin

Myosin

filament

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-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 mechanical strength to the cytoskeleton – specialized for bearing tension

(like microfilaments)2. support cell shape

e.g. forms the axons of neurons3. anchor & stabilize organelles

e.g. anchors the nucleus in place4. transport materials

e.g. movement of neurotrasmitters into the axon terminals

3. microtubules = hollow rods or “straws” - 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

-MT disassembly is a mechanism of certain chemotherapy drugs

http://www.nature.com/nrc/journal/v4/n4/fig_tab/nrc1317_F4.html

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

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 have a role in mitosis - spindle and chromosome alignment

Non-membranous Organelles

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

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 microtubuledoublet

Dynein proteins

Centralmicrotubule

Radialspoke

Cross-linkingproteins betweenouter doublets

Plasma membrane

Triplet

(c) Cross section ofbasal body

•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− 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

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

1

2

3

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

Membranous Organelles

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

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

The Endomembrane System: A Review• endomembrane system is a

complex and dynamic player in the cell’s compartmental organization

• divides the cell into compartments

• includes the:– Nucleus– Endoplasmic Reticulum – Golgi apparatus– lysosomes, endosomes– vacuoles and vesicles

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

1. 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

-three functions: 1. synthesis – phospholipids, lipids and proteins•proteins •phospholipids & lipids

•2. storage – intracellular calcium

•3. transport – site of transport vesicle production

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

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

Co-translational Protein Synthesis

two kinds of proteins enter the ER:1.ER proteins – transmembrane proteins that stay stuck in the ER membrane PLUS ER lumen proteins that remain in the ER2. proteins destined for the Golgi, PM or lysosome or secretion

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)

• 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

Co-translational Protein Synthesis

Translocation• try this animation – it might be a bit complicated – but give it a

try anyway• http://www.rockefeller.edu/pubinfo/proteintarget.html• here’s a figure from a molecular biology text that summarizes

the process

• 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

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

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/webcontent/animations/content/proteinsecretion_mb.html

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 madein the ER undergo N-linked glycosylation

Smooth ER – extends from the RER-free of ribosomesmain function is transport vesicle synthesis – area where this happens can be called transitional ER

1. Endoplasmic reticulum (ER)

-but other cell types have SER with enzymes embedded in it for additional functions: 1.lipid and steroid biosynthesis for membranes2. detoxification of toxins and drugs3. cleaves glucose so it can be released into the bloodstream4. uptake and storage of calcium

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

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

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

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

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

Proteoglycan

• 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

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

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)

some PM proteins and most secretory proteins are synthesized as larger, inactive pro-proteins that will require additional processing to become activethis 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)

Modifications in the Golgi: Protein Trimming

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 Golgi2. 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

targets:1. secretory vesicles for

exocytosis2. membrane vesicles for

incorporation into PM3. transport vesicles for the

lysosome

SO - WHERE DO PROTEINS GO AFTER THE SO - WHERE DO PROTEINS GO AFTER THE GOLGI???GOLGI???

-proteins budding off the Golgi have three targets:

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

WHAT IF YOU ARENWHAT IF YOU AREN’’T ONE OF THESE PROTEINS??T ONE OF THESE PROTEINS??

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

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

-acidic 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)

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

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 ofnervous function-most common in Ashkenazi Jews, FrenchCanadians and Cajun populations in Lousiana (same mutation as Jews)

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 intofolds called cristae -these increase the membrane surface area for the enzymes of Oxidative Phosphorylation

•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 Kreb’s Cycle)

Cellular Respiration

http://biology.about.com/gi/dynamic/offsite.htm?site=http://www.sp.uconn.edu/%7Eterry/images/anim/ETS.htmlhttp://biology.about.com/gi/dynamic/offsite.htm?site=http://www.biocarta.com/pathfiles/krebPathway.asphttp://vcell.ndsu.nodak.edu/animations/etc/movie.htm

-glycolysis-transition phase-citric acid cycle-electron transport chain

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 Kreb’s 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)

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 oxygenPROBLEM #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:-other functions of peroxisomes:1. synthesis of bile acids1. synthesis of bile acids2. breakdown of alcohol by liver cells2. breakdown of alcohol by liver cells

Adrenoleukodystrophy and peroxisomes: -X linked disorder in the gene ABCD1 (transporter protein)-1:20,000 to 1:50,000 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 oilLorenzo Odone = “Lorenzo’s Oil” (mixture of unsaturated fatty acids

that slows the developmentof these saturated FAs)

F-actin and peroxisomes