L1-ReviewSubcellular

Introduction to Cell Structure The basic unit of life is the cell. Cells can exist as individual units (e.g. leukocytes) or they can be grouped together to form tissues. Tissues make up organs, which in turn form organ systems.

Gartner & Hyatt, 2009

Visible light is passed through specimen, collected through a series of lenses that magnifies the image, and the image viewed through oculars. Effective magnification is about 1000 x, and limit of resolution is 0.2 m.

Methods to Examine Cells and their OrganellesLight microscopy - Immunofluorescence

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yantingwangTypewritten Texthe resolution of a microscope defines its ability to differentiate two objects when you view them on a specimen slide.

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Require initial fixation of cells or tissue Direct immunofluorescence: the 1 Ab is directly labeled with the fluorophore Indirect immuno-fluorescence: the 2 Ab is labeled with the fluorophore.







Fluorescent substances absorb light at one wavelength and then emit light of a longer wavelength.





A method widely used in diagnostic labs is staining of tissues with H&E Hematoxylin positively charged binds to negatively charged cell components such as heterochromatin, nucleoli, and rough ER stains them blue/purple Eosin negatively charged binds to positively charged components such as proteins in cytoplasm and extracellular matrix stains them red/bright pink

Methods to Examine Cells and their OrganellesLight microscopy Hematoxylin & Eosin (H&E)

Fix tissue

Dehydrate embed in wax

Section tissue

Remove wax with xylene &

alcohol

Stain with H&E

Methods to Examine Cells and their OrganellesLight microscopy Hematoxylin & Eosin (H&E)

H&E stain of nerve cells

H&E and TEM of adipocyte

Ovalle, 2008 Ovalle, 2008

In principal, transmission electron microscopy (TEM) works in a similar manner to the light microscope. The electron microscope has a theoretical resolving power of 0.1 nm and can achieve magnifications > 100,000 x. An alternative form of EM is scanning electron microscopy (SEM), where an electron beam is scanned over the surface of the tissue.

Methods to Examine Cells and their OrganellesElectron microscopy TEM and SEM




Methods to Examine Cells and their OrganellesLight and Electron microscopy comparison

H&E TEM SEM

Chondrocytes prepared three ways

Ovalle, 2008 Ovalle, 2008 Ovalle, 2008

Cellular OrganellesNucleus morphology The DNA of eucaryotic cells is sequestered in the nucleus, which is enclosed by the nuclear envelope. The nuclear envelope consists of two membrane bilayers: an outer membrane that is contiguous with the ER and an inner membrane. The latter is associated with a network of intermediate filaments called the nuclear lamina. Fawcett, 1966






yantingwangTypewritten Textmay be studdedwith ribosomes


yantingwangTypewritten Textlies underneath nuclear membraneserves as a scaffold for attachment of chromatin

yantingwangTypewritten Textnucleus is often ovoid but in some cells MULTIOBULAR some contain MULTIPLE NUCLEUS (osteoblasts, straited muscle cells

The nuclear envelope is punctuated at intervals by nuclear pores, which regulate protein and nucleic acid transport into and out of the nucleus. Major function of the nucleus is to contain the chromatin and regulate the entry and exit of macromolecules between the cytoplasm and nucleoplasm.

Cellular OrganellesNucleus pores and function

Fawcett, 1966






Cellular OrganellesNucleus chromatin Heterochromatin condensed chromatin that is electron dense and is not transcriptionally active Euchromatin uncoiled DNA that is electron lucent and is transcriptionally active Nucleolus site of rRNA transcription and ribosome assembly

Fawcett, 1966










ER - ERGIC- cis Golgi - medial Golgi - trans Golgi - TGN - PM

Cellular OrganellesOverview of Secretory Pathway

Gartner, 1997


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ER consists of flattened sacs (cisternae) and tubulovesicular elements connected into a network (reticulum). Some regions of the ER are studded with ribosomes (rough ER) whereas others are not (smooth ER). Rough ER critical for secretion and synthesis of membrane proteins. Smooth ER is sight of lipid synthesis and is also a reserve of Ca2+ ions that are released in response to second messengers.

Cellular OrganellesEndoplasmic reticulum organization and function

Ovalle, 2008








yantingwangTypewritten TextRER: make intergral and solublle proteins

yantingwangTypewritten TextSER: lipid and carb metabolismCA+2 reservoir for 2nd messenger

Cellular OrganellesEndoplasmic reticulum TEM

Fawcett, 1966 Fawcett, 1966

The intermediate compartment/ ERGIC (ER-Golgi-intermediate compartment)/ CGN (cis-Golgi network)/VTC (vesiculo-tubular clusters) serves as a way-station between the ER and Golgi. COPI-mediated sorting events, such as retrieval of escaped ER-resident proteins occur in this compartment.

Cellular OrganellesIntermediate compartment




yantingwangTypewritten TextDifferent namesERGICCGNVTC

The Golgi complex consists of s t acks o f f l a t t ened sacs (cisternae) and associated vesicular elements. The cisternae are arranged into four distinct compart-ments: cis-Golgi, medial-Golgi, trans-Golgi and trans-Golgi network (TGN), each with a different composition and function. Stacks associate to form a ribbon-like structure.

Cellular OrganellesGolgi complex










The Golgi is juxtanuclear and held there via microtubule attachments.

Lipids and proteins from the ER are transported vectorially through the Golgi, then sorted in the TGN for delivery to their final destinations.

Site of numerous posttranslational modifications including: maturation of N-linked and O-linked oligosaccharides, proteolysis, lipid attachment to proteins, sulfation, and phosphorylation.

Golgi

ER

Cellular OrganellesGolgi complex - function

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yantingwangTypewritten Textcis-->medial-->trans (sorting)


yantingwangTypewritten Textduring transit thru golgi complex, many proteins and lipids are posttranslationally modifiedincludes proteolytic processing

Cellular OrganellesGolgi complex - TEM

Fawcett, 1966 Fawcett, 1966

Ovalle, 2008


The plasma membrane is a single bilayer that surrounds and protects each cell. Regions of the plasma membrane are specialized for adhesion to the substratum or extracellular matrix and for adhesion to and communication with other cells. In addition, the plasma membrane contains numerous receptors that can respond to external signals and trigger intracellular production of second messengers in response to physiological cues.










The plasma membrane can have specialized projections including: M i c r o v i l l i a c t i n - b a s e d structures that increase the surface area of the plasma membrane. Ci l ia microtubule-based structures that are important for motility and sensory perception S te reoc i l i a ac t in -based structures that are critical for hearing


Fawcett, 1966

Ovalle, 2008

Ovalle, 2008








Cellular OrganellesEndosomes

yantingwangTypewritten Text1. recieve and sort fluit and membraneinternalized from cell

yantingwangTypewritten Text2. much of the material is recycled BACK into the Plasma Membrane



yantingwangTypewritten Text3. Remainder is delevered to Late endosomethen to Lysosome




yantingwangTypewritten TextBECOME MORE ACIDIC as they "mature"



yantingwangTypewritten TextNOTE: endocytic and biosynthetic pathway are not completelydistinctsome endocytosed protein is delevered to TGNbefore being recycled to cell surfaceSome nearly synthesized proteins pass thru endosomes before reaching cell surface









Endosomes are a heterogeneous population of tubules and vesicles that mediate transport of internalized proteins and lipids. Endosomes can be divided into early and late populations: early endosomes receive and sort fluid and membrane internalized from the cell surface; Much of this material is recycled back to the plasma membrane, while the remainder is delivered first to late endosomes and then to lysosomes. The endocytic and biosynthetic pathways are not completely distinct as some newly-synthesized proteins enter endosomes before reaching the cell surface.

Cellular OrganellesEndosomes

Cellular OrganellesEndosomes light micrograph and TEM

Early endosome nucleus

Lysosomes are acidic (pH ~ 5), vesicular structures that contain ~ 50 hydrolytic enzymes. By EM they appear dense and heterogeneous and sometimes contain multiple vesicles. Proteins, carbohydrates, and lipids are metabolized in lysosomes and the resulting amino acids, sugars, and nucleotides are released into the cytosol for reutilization.

Cellular OrganellesLysosomes









Cellular OrganellesLysosomes - TEM

Ovalle, 2008

Mitochondria are bound by a double bilayer that creates four compart-ments: the outer membrane the inner membrane (which is thrown into folds called cristae) the in termembrane space the matrix

Cellular OrganellesMitochondria

Ovalle, 2008




Mitochondria are motile organelles, and actively fuse and divide within cells. A critical function of mitochondria is to synthesize ATP and NADH for the cell using energy derived from electron transport and oxidative phosphorylation.

Cellular OrganellesMitochondria

mitochondria microtubules





yantingwangTypewritten Textthought to have evolved from bacteria that were engulfed by other cellsas a result, contain a small genome that encodes for few proteinsproteins localized to IMM

Cellular OrganellesMitochondria SEM and TEM

Ovalle, 2008

Ovalle, 2008 Fawcett, 1966

P e r o x i s o m e s a r e r o u n d , membrane-bound organelles that contain enzymes that produce hydrogen peroxide as a byproduct of oxidative reactions. They are important in the breakdown of fatty acids and in the detoxification of various molecules. Some of these enzymes are p r e s e n t a t s u c h h i g h concent ra t ions they form electron-dense paracrystalline arrays.

Cellular OrganellesPeroxisomes

Ovalle, 2008









Cellular OrganellesCytoskeleton

g-actin

microtubules actin

Cellular OrganellesCytoskeleton - IF

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yantingwangTypewritten Textplay a major role in organizing the structure and activity of the cellMOST IMPORTANT F(X) is to provide mechanical support-also involved in motility (Xsome movement during cell divisioncell locomotionbeeting of cilia and flagellavescile traffickingphagocytosisComprised of 3 DIFFERENT FILAMENT systemsactin (microfilament)intermediate filamentmicrotubutes

yantingwangTypewritten Textcomprised of 2 strands of F-ACTIN intertwinedto from ALPHA HELIX-diameter of fiber is 4-7nm-

yantingwangTypewritten Text(MICROFILAMENTS)

yantingwangTypewritten TextINTERMEDIATE FILAMENTassemblages of fibrous protein coiled into thicker cables8-12 nm thick-many typesnuclear laminsvimentin (mesenchmal orgin)demin (myscke cellglial fibrarlly acid proteinperipherincytokeratininsnuerofillaments




yantingwangTypewritten Textcomprised of 13 columns of alphaand beta tubulinsform hollow tube25nm diameter

Cell Biology is Fun, Colorful and Clinically Relevant!

L1-ReviewSubcellular

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