Fundamentals I: 10:00 - 11:00 Scribe: Christopher Bannon

Monday, August 10, 2009 Proof: NA

Dr. Cotlin Cell Organelles and Ultrastructure I Page 5 of 6

PM – Plasma Membrane, RBC – Red Blood Cells, ER – Endoplasmic Reticulum, RER – Rough ER, ECM =Extra Cellular Matrix, SRP = Signal Recognition Particle, EM=Electron Micrograph

I. Cell Organelles and Ultrastructure [S1]:

a. Need to know how individual cells of body work before can understand body in terms of systems

II. Lymphocyte v Neuron [S2]

a. –Gene expression = fundamental difference between cells

i. Expression dictates structure, function, and differentiation

b. Same DNA compliment in all cells;

i. Exception = RBC which loses its nucleus

ii. Only difference among cells are which genes are expressed

1. Gene expression brings compliment of proteins which dictate structure & function

III. Organelle Classification [S3]

a. Membrane Bound

i. PM composed of double phospholipid bi-layer

ii. Nucleus = double membrane bound (folded back on itself but is same membrane)

iii. RER & SER, Golgi, lysosome, endosomes, peroxisomes[ all membrane bound], mitochondria (2 membrane systems ~ nucleus)

b. Non-membrane bound/ found freely floating in cytoplasm

1. Ribosomes,

2. proteosome,

3. cytoskeletal elements:

a. microtubules,

b. intermediate filaments,

c. microfilaments,

4. Centrioles

5. Cilia & Flagella

6. Random Inclusion

a. Pigments

b. Crystals

IV. Schematic of a Polarized Cell [S4]

a. Polarized (2 different ends; positive and neg charge on proteins)

i. Cell has distinct apical and baso-lateral region

1. Its an epithelial cell with microvilli at top, most likely from GI tract

b. Observation reveals, nuclear membrane is continues with ER membrane

i. Degree of prevalence of organelles dictated by cell function

1. Excretory cells (in salivary gland) = lots of ER b/c need to produce lots of proteins

2. Macrophages enriched in lysosomes

V. Functions of the Cell Membranes [S5]

a. Maintain cell structure

b. Control all movement

c. Regulation of particles entering /leaving cell

d. Interactions

i. Cell-cell

ii. Cell-matrix

e. Receptors

f. Transport

g. Signalling

VI. Molecular Composition of the Cell Membranes [S6]

a. Lipid

i. 2 fluid layers of phospholipids (negatively charged phosphate group on end of hydrocarbon chain)

ii. Glycolipids (have attached sugars

iii. Cholesterol (sterol derivative)

iv. All polar, “amphipathic”

b. Protein

i. Integral (physically spanning bi-layer)

1. Transmembrane

2. Multipass

ii. Peripheral (associated with outside of membrane/ do not pass through)

VII. Fluid Mosaic Model of the Cell Membranes [S7]

a. Example of integral membrane proteins (mutli-helical proteins completely spans bi-layer)

i. Some part of integral is physically incorporated into membrane

b. Peripheral proteins = NOT physically bound or integrated, just associated with it

i. Can allow for transient interactions with membrane

VIII. EM of Cell Membrane[S8]

a. Each cross section is an example of a microvilli from the polarized GI cell mentioned earlier

b. EM = electron micrograph

IX. Two Important Concepts about Membranes [S9]

a. Nothing is static

i. , proteins freely move throughout the membrane; nothing is anchored; fluid bi-layer.

ii. However there are large chunks/ lipid rafts, or things tethered together and move in unison;

1. Group of proteins involved in signaling to maintain the messenger chain of events.

2. Some are grouped together/ tethered through disulfides and other linkages/ some are free floating

iii. As seen on left, stars =labeling proteins; when 2 membranes join eventually proteins will disperse in new PM evenly (thermodynamic law)

iv. Proteins & phospholipids are highly mobile

b. Arrangement (distinct orientation)

i. Cytosolic Face

ii. Extracellular Face

1. Glycoproteins (carbohydrate modification always on extracellular side)

2. Organelles have cytoplasmic and luminal side (inside organelle)

a. Sometimes modifications only on 1 face

3. Possible different compliment of phospholipids on extracellular vs. intracellular sheet of bi-layer

X. Functions of Integral Membrane Proteins[S10]

a. Channels & pumps for ions

i. Allow passage for only 1 variety of ion (Na, Ca, K, etc);

ii. Ca & K are selective and can be gated

iii. Ions don’t bind

iv. Ions move down concentration gradient(driving force)

b. Structural,

i. internal structural = linkers,

ii. receptors = take up and bind signaling molecules

c. Pumps/ Carriers

i. Physically bind

ii. Require energy for conformational change

iii. Na/K, glucose

d. Receptors

i. Receptor mediated endocytosis

1. LDL (low density lipoprotein) enters cell through LDL receptor

2. Transferon (iron transporter)

3. Growth Factors

e. Signaling molecules

i. Kinases (adds phosphate to molecule)

ii. Phosphatases (remove phosphate)

iii. Steroid receptors

f. Linkers (all cells byt RBC’s)

i. Link to other cells or the matrix

ii. Proteins on surface of cells link to surface proteins of other cells

XI. Channel & Pump/Carrier Proteins[S11]

a. –

XII. Surface Receptors & Linkers and Structural Proteins[S12]

a. –

XIII. Structure of the Nucleus[S13]

a. Double membrane (same membrane folded on itself= Nuclear Envelope)

i. Outer membrane of nuclear envelope = continuous with ER membrane

ii. Has nuclear pores = tight regulation of particles entering and exiting nucleus

iii. Intermediate filaments = give meshwork support surrounding nucleus

b. Condensed Chromatin (heterochromatin = blue)

c. Loose Chromatin (euchromatin = black coils inside the nucleolus)?????

d. Nucleoulus

e. Spaces/ gated openings = nuclear pores

XIV. EM of the Cell Nucleus [S14]

XV. The Secretory Pathway [S15]

a. ALL proteins destined for secretory pathway

i. ER,

ii. Vesicles

iii. , Golgi

1. cis=receive contents from ER,

2. medial

3. trans=where small secretory vesicles bud off & travel to PM

iv. Endosomes= vesicles that take in cargo from ECM

1. Early = newly broken off of PM

2. Late = those traveling to Golgi or to Lysosomes

v. Lysosome = degradative organell

b. Any contents in secretory vesicles arose from ER

c. Ribosome/ Actin molecules NEVER associated with secretory pathway

XVI. The Endoplasmic Reticulum [S16]

a. Flattened Stacks = sites of membrane & secretory protein synthesis

b. RER has ribosomes (transiently attached, not permanent; ER not have attached ribosomes

c. Meteabolize Drugs,

d. Modifiy proteins

i. Disulfide bonds formed

ii. Glycosilation of proteins

e. Vesicles always from ER to golgi and also coming from cytoplasm to ER to prevent ER depletion

i. Same for PM, can’t deplete phospholipids or proteins

ii. Nothing is Stagnant

XVII. RER [S17]

a. Membrane of it is studded with ribosomes

XVIII. Major Locations of Protein Synthesis [S18]

a. All protein synthesis occurs in cytosol

i. mRNA transcribed in Nucleusè then travel to cytoplasm where ribosome binds to 5’ end and transcription occurs

XIX. Free & Membrane Bound Ribosomes [S19]

a. All ribosomes are the same (no diff between cytosolic and ER bound ribosomes)

XX. Ribosomal Attachment and Transcription on ER [S20]

a. mRNA binds to ribosome and transcription occurs

i. Protein’s for secretory pathway have signal for ER that exists in transcribed portion

ii. Receptor for signal binds to it and transports it ER where binding occurs

b. Those lacking messenger stay in cytosol

c. Free ribosome = exists in cytosol and has no affiliation with ER

d. Polyribosome = when subsequent ribosomes bind to mRNA once the previous ribosome has finished transcribing a set length of the mRNA

i. Lots of glycolitic enzymes use this to increase efficiency

XXI. Protein Transcription [S21]

a. Ribosomes bound to ER (not a permenant association)

b. Free ribosome in cytosol transcribe proteins for: Nucleus, Mitochondria, Chloroplast, & Peroxisomes

i. Distinct signals on fully transcribed protein that take protein directly to destination (exclusive of secretory pathway)

c. Those in secretory pathwayè small portion transcribedè SRP binds to protein fragment NOT to ribosome ( protein is still associated with ribosome)è SRP binds to SRP receptor on ER membrane such that protein is transcribed and enters into Translocation complex in ER membrane passing completed peptide into ER lumenè destined for rest of secretory pathway

d. Ribosome dissociates with protein and mRNA once transcription completes

e. Student Question: Is it not attached to microbutles or anything/ directed?

i. Answer: NO, not at this point, b/c if a ribosome is making a glycolitic enzyme, it will make it and keep making it and stay in the cytosol. If ribosome is making a peptide for secretory pathway, peptide will have a signal sequence and the sequence will bind to SRP. Microtubules are implicated in moving of vesicles, from golgi out to PM or across cells, but proteins not for secretory pathway are synthesized and stay in cytosol close to where it will be used (ex glycolitic protein already near required location), therefore microtubules are not required to transport protein a long distance across the cell.

f. Amino group of protein can be in cytosol, Carboxyl group sometimes in lumen/extracellular; sometimes flipped

XXII. ER and Golgi Apparatus [S22]

a. ER is continuous with nucleus not with Golgiè Proteins of ER bud off and fuse with Golgi on cis face

b. In Golgi

XXIII. ER and Golgi [S23]

a. Picture of vesicle budding off ER in direction of Golgi

XXIV. The Golgi Complex [S24]

a. Vesicles received on cis and leave Golgi on trans face–

b. Terminal/final glycosylation of peptides occurs in Golgi,

i. Initial glycosylation can start in ER

XXV. What Happens in the Golgi [S25]

a. Sorting occurs in Golgi

i. Vesicle budding off Golgi, & has no signal è goes to fuse with PM

ii. Go to lysosome, ER has distinct signal for desired location

iii. Post-translational modifications occur

1. Sugars added

2. Di-sulfide linkages

3. Cleavage of proteins

iv. Trans face is the sorting site for outwards flow (from Golgi)

b. Bi-directional flow, from lysosome to Golgi for example

XXVI. Dyed Picture of Golgi [S26]

a. Cis face is the smaller side

b. Trans face = large side

XXVII. The Lysosome [S27]

a. Contains degradative enzymes (enzymes go through Secretory pathway

i. From ERè Golgiè has modified sugar Mannose 6-phosphate = this a marker for protein to be transported to lysosome

XXVIII. Lysosomes (diagram) [S28]

a. All degradative enzymes present in lysosome lumen

i. Proteases= break down proteins

ii. Lipases= break down lipids

iii. Nucleases = break down nucleic acids

iv. Phosphatases

v. Aryl sulphatases

b. Lysosome pH =5.5, acidic environment required for enzymes to be catalytically active

i. Protective mechanism incase lysosome bursts, pH of cytosol will quench enzymatic activity (cytosol~7+/-) so don’t break apart cell

XXIX. Lysosomes (photo) [S29]

a. Appear as electron dense vesicles in EM; dark vesicles

XXX. Endocytosis (photo) [S30]

a. “Opposite” of secretory vesicle as, endosomes bud from PM and travel into cell (lysosome),

b. Receptor mediated endocytosis

i. Upon binding, budding proceeds and travels into cell

XXXI. Phagocytosis (photo) [S32]

a. Process of taking up large molecules; function of macrophages

b. Large scale of endocytosis

XXXII. Peroxisomes [S33]

a. Not associated with secretory pathway

b. Proteins for peroxisomes= synthesized in cytosol then specific signals on protein allow for transport to peroxisomes

c. Peroxisomes =oxidizing environment (fatty acids broken down but NOT for energy)

d. Used to clear debris from cytosol

XXXIII. Peroxisomes (photo) [S34]

a. Lysosome takes care of elements needed to be degraded that are in secretory pathway

b. Peroxisomes are smaller than lysosomes

XXXIV. Mitochondria (photo) [S35]

a. Site of ATP generation

b. High prevalence in muscle and nerve tissue

XXXV. Organization of Mitochondria [S36]

a. Divide via binary fission

b. In Cell division, cell makes extra golgi or ER to prepare for mitosis

c. Mitochondria proliferation NOT connected to cell division

XXXVI. Overview of Mitochondrial Function [S37]

a. Have own DNA, allows for encoding some of own proteins; other proteins must be synthesized in cytosol and transported into mitochondria

i. Own ribosomal machinery

b. Have own protein processing machinery

c. Outer membrane is continuous

i. Very permeable

d. Inner membrane=highly folded (folds = called cristae)

i. Where components for electron transport complexes and ATP synthase are imbedded

ii. Very impermeably; highly regulated

e. Inter-membrane space (yellow on slide

f. Internal region = matrix

g. Slide lists all relevant metabolic processes such as TCA (the citric acid) cycle

XXXVII. Mitochondria (photo) [S38]

a. Proliferation when metabolic demands are high

XXXVIII. Inclusions [S39]

a. Observable in cytosol

b. Liver stores glycogenè regulate blood glucose levels

c. Lipfuscin= age related pigment accumulating in cells overtime

d. Pigments

e. Crystal

XXXIX. Non-membrane Organelles [S40]

XL. Ribosomes [S41]

a. All consist of large and small subunit (they bind during translation)

b. Complex of rRNA and ribosomal proteins

i. rRNA is always associated with ribosome

ii. associate with mRNA

iii. make many proteins/ used many times (recycled)

XLI. Major Locations of Protein Synthesis [S42]

XLII. Proteosome [S43]

a. Involved in degradation of proteins in cell

b. Composed of Cap and tubular Core

i. When protein in cytosol needs to be degraded (finished use/ function), Ubiquitin (small peptide tag) will be placed on any protein that is to be degraded by proteosome

c. Full of proteases

XLIII. Cytoskeletal Elements [S44]

a. Actin filaments aka Microfilaments

b. All structures are polymers of small subunits (be they monomers or dimmers)

XLIV. Cytoskeletal Elements Cont’d [S45]

a. Actin/ Microfilaments

i. Polymer of actin monomer

ii. g-Actin = globular actin = free actin molecule

iii. f-Actin = filament actin = polymerized into long strand

b. Microtubules

i. Polymerized from Tubulin dimers (alpha & beta)

ii. Polymerize by non-covalent means

1. Associate end to end and side to size = hollow tube

c. Intermediate Filaments

i. Individual monomers = long fibrous subunits (analogous to nylon rope)