3 - Cell Biology (ER, Golgi, and Vesicles)

Golgi Apparatus staining

****golgi does not stain!!!! it appears as a clear negative space hof regions (free space)

Lipid Inclusions

Adipocytes (fat cells) contain one massive lipid inclusion. The nucleus is squeezed into a corner and the cytoplasm is just a thin rim beneath the plasma membrane. Since the lipids are usually extracted in processing the slide, they appear as clear spaces In contrast, the smaller, multiple cholesterol-filled inclusions in these steroid producing cells, just give the cytoplasm a bubbly (flocculent) appearance.

Inclusions

Inclusions are generally not membrane bound and are not metabolically active. Glycogen: In tEMs this sugar forms small dark patches of particles. Lipids (e.g. cholesterol): these form multiple small droplets in many cells. They are more common in steroid producing cells. Fat cells contain a single, very large inclusion. Glycogen Granules Peroxysome Junquiera tEM Other inclusions are breakdown products of cell metabolism that are difficult to remove: Lipofuscin: a breakdown product commonly found in neurons, is membrane bound, breaking the rule stated above.

sER under the microscope

ONLY SEEN IN tEM not visible in LM In the case of steroid producing cells that have lots of sER and lipid inclusions, you can see the inclusions as bubbles in the cytoplasm

How are the Golgi secretory vesicles regulated?

The targeting of the secretory vesicles budding off the trans side of the Golgi apparatus is also controlled by membrane associated proteins. These include: clathrin, AP-3, and caveolin "traffic directors" *****Vesicles without trafficking signals are constitutively secreted- their products exit the cell as fast as they are made. (the Vesicle and membrane fusion are not regulated, their exit is continuous as they are made)

trans-face of golgi apparatus

The trans-face is the maturing face. On this side, secretory vesicles bud from the Golgi apparatus. The stacked elements of the Golgi "transport" side of the golgi Usually, the trans-face is concave

Pinocytosis via Clathrin-Coated Pits

One of the most important mechanisms for endocytosis is through the use of clathrin-coated pits. This process is commonly used for receptor mediated endocytosis. Step 1. The material the cell wants from the extracellular fluid (Example- LDL) is attached to the surface via receptors. Step 2. The receptors activate clathrin attached to the cytoplasmic surface of the membrane to form a coated vesicle. 3. The clathrin disengages from the vesicle and returns to the plasma membrane to be used again. 4. The vesicle divides into a return vesicle that brings most of the plasma membrane and the receptors back to the surface for reuse, and an endosome containing the needed material. Optional Step 5. If further degradation is needed, the endosome fuses with a lysosome.

Caveolin

directs them to a pool near the surface of the cell (apical surface). they will collect there until secretion is intiniated.

AP3

directs them to endosomes

Clathrin

directs transfer vesicles to endosomes and lysosomes

****How can you tell the lysosomes from phagosomes?

Phagosomes under tEM will appear to have a heterogenous contrasting content. Lysosome will have one color content.

Where does protein production happen?

in the cytoplasm

Clathrin Actions

Plasma membrane does not normally like to bend sharply. Clathrin molecules, which have an unusual triskelion shape, are able to attach to the plasma membrane and bend it sharply when they receive the appropriate signal. This allows them to form a coated pit. Once the pit is fully formed, another molecule, dynamin, forms a collar that can then snip off the neck of the vesicle, forming an endosome.

Telling Glycogen Inclusions & Polyribosomes Apart

Polyribosomes are smaller, more organized and less dense than glycogen granules

*****Ribosome protein target

Polyribosomes: proteins are designated for the cytoplasm, nucleoplasm (of the nucleus), mitochondria, and peroxisomes rER: proteins designated for lysosomes, for secretion, and within membrane bound organelles.

*****KNOW THESE FILAMENT TYPES, CELL TYPES, AND FUNCTIONS

keratin desmin tonofilament vimentin glial fibrillary acidic protein (GFAP) neurofilaments nuclear lamins

Motor proteins

The microtubules provide tracks for moving organelles and other cargo around the cell. The microtubules just provide the pathways. The muscle is provided by two motor proteins: Dynein and Kinesin. Dynein moves cargo toward the Minus end and the centrosome. Kinesin moves cargo toward the Plus end, away from the centrosome. Both processes are powered by ATP. DYNEIN ---> (-) minus end KINESIN ---> (+) plus end

Smooth Endoplasmic Reticulum (sER)

The sER shares a role in membrane phospholipid synthesis with the rER Most cells have a relatively small amount of smooth Endoplamic Reticulum (sER). A few cell types are exceptions to this and have lots of sER: • Cells that synthesize steroids from cholesterol (e.g., interstitial cells of Leydig that produce testosterone) • Cells that detoxify materials (e.g., liver cells that deal with barbiturates, etc.) • Skeletal muscle cells (in this case, the extensive sER sequesters calcium ions. It is so specialized it has a special name: sarcoplasmic reticulum)

tEM of rER, sER, and Golgi Apparatus

rER -- contains ribosomes "specks" sER - round and continuous with the nucleus Golgi - looks like pancakes

***Where is each component produced? rRNA tRNA mRNA

rRNA -- produced in the nucleolus tRNA -- produced in the nucleus mRNA -- produced in the nucleus **these two are produced in the nucleus and transported out to the cytoplasm via nuclear pore

Phagocytosis is a multistep process

1. Recognition & Attachment: In the first step, receptors on the surface of the cell recognize and stabilize the item that will be phagocytoses 2. Engulfment: In the second step, part of the cytoskeleton (actin filaments) beneath the plasma membrane extends plasma membrane coated arms out to surround the targeted item, forming a phagosome. 3. Lysosomal Fusion: In the third step, lysosomes formed in the Golgi apparatus fuse with the phagosome, forming a phagolysosome. 4. Degradation: The lysosomal enzymes degrade the item into its constituent parts. Some of these are used by the cell, the rest remain in a secondary lysosome.

****How do you know if a protein is made by a polyribosome or by rER?

1. The key is the 5' end of the mRNA. 2. If a signal peptide sequence is present, then the forming polypeptide will bind a signal receptor protein (SRP). 3. The SRP binds to an SRP receptor on the rER membrane. 4. This causes the SRP to disengage, freeing the polypeptide to thread through a channel protein called a Translocon. 5. Attached to the translocon is a signal peptidase, which cuts off the no longer needed signal peptide. Now as the polypeptide is made, it moves into the cistern of the rER Membrane proteins have start and stop sequences that cause some of the protein to be located in the membrane, with other parts inside or outside the membrane

**What is the function of sER?

1. synthesize phospholipid membrane 2. synthesize steroids from cholesterol 3. detoxify materials (liver cells that deal with barbituates) 4. sequester calcium in the muscle cells (sarcoplasmic reticulum)

Actin & Myosin

Actin also has a motor protein associated with it: Myosin. As shown in the fluorescence image, microfilaments are present throughout the cell and can be used by individual myosin molecules as a framework for moving cargo. Alternatively, the myosin can also be used to rearrange the microfilaments. As with the other motor molecules, hydrolysis of ATP provides the energy source for the movement.

Cytokinesis

Another important role for microtubules and centrosomes is in cytokinesis (cell division). They form the spindle apparatus. During this process the centrioles duplicate and move to either side of the cell. The chromosomes also duplicate. They line up in the metaphase plate. Then they are pulled apart by the microtubules during the process of cell division.

Cilia and Axonemes

Another place microtubules are found is within clia. Cilia are extensions of the plasma membrane that contain an array of microtubules called an axoneme. The axoneme is made up of nine microtubule doublets, where the heterodimers are shared, plus a central pair of microtubules, and additional connecting protein elements. Each doublet is connected to an adjacent doublet by a pair of dynein arms. The dynein arms can move when energy is supplied by ATP. This bends the axoneme and moves the entire cilia. The axoneme grows from a basal body that is very similar to a centriole in its construction in that it is made up of microtubule triplets.

Lysosomal contents

As can be seen by the long list of enzymes contained in lysosomes (which you do not have to memorize), they have all the tools necessary to chew up most biological materials. Due to the presence of an ATP powered proton pump in the lysosomal membrane, lysosomes have a very low pH. This acidic environment also helps degrade materials in the lysosome. More importantly, all the lysosomal enzymes are only active at this low pH. So if a lysosome is damaged, and these enzymes leak out, they are inactive in the neutral pH of the cytoplasm Lysosomes can be used to digest material ingested into the cell. They are also used to digest and recycle cellular organelles that are worn out.

****what is the significant of lipid inclusions when it comes to microbes?

Bacteria & Viruses attempt to utilize lipid inclusion for their own energy, but inclusions often contain anti-bacterial and anti-viral compounds to thwart this.

Intermediate Filaments Types

Different cell types have different intermediate filaments in them. This is important for pathology. Often one can identify the origin of a tumor by using antibodies to these different intermediate filament types. Cancers of different types require different treatments. Pic: Here the antibody to desmin is attached to a green fluorescent marker, showing the intermediate filaments in this smooth muscle cell

Lysosomal Storage Diseases

If there is something wrong with one of the lysosomal enzymes due to a genetic anomaly, often the results are very bad. The material that can no longer be broken down by the cell builds up, eventually disabling or killing the cell. Babies born with many of these diseases have very short, uncomfortable life spans.

Protein production: In polyribosomes (rosettes)

In Polyribosomes: Polyribosome rosettes are produced when a series of ribosomes are lined up along a single strand of mRNA. The ribosomes move along the mRNA, each translating it into a protein strand.

Protein production: In rough ER

In Rough ER: The proteins manufactured by ribosomes on rER are inserted into the cistern of the endoplasmic reticulum, or are inserted into its membrane. (blue) red arrow -- polyribosomes (rosettes)

rER staining properties

In secretory cells, the abundant rER is organized into stacks. The large amounts of rER present in secretory cells gives their cytoplasm a purple cast in H&Es because the basophilic RNA attracts hemotoxylin.

Intermediate Filaments construction

Intermediate filaments are constructed from subunits that are polar coiled-coil dimers with a head and a tail. These are stacked together head to tail, but other subunits are stacked alongside in a staggered fashion to create an apolar protofilament. Protofilaments are bundled together to make a protofibril and these are then tied together to make the actual intermediate filament, which is about 10 nm in diameter.

***How can you tell secretory vesicles from lysosomes? (in tEM)

It is difficult to tell secretory vesicles (left) from lysosomes (right), unless the lysosomes have become phagosomes and have partially digested elements in them. However, secretory vesicles often collect near the apex of polarized cells. This is not true of lysosomes. Autophagosome containing senescent mitochondria and ribosomes

Microfilaments (AKA Actin)

Microfilaments are composed of actin monomers arranges as dual helical strands. So they are also called actin fibers. The actin monomers are added to the plus end and removed from the minus end (conveniently). If the rates are the same, this results in monomer turnover, but no change in the length of the fiber. This is called "tread milling". If there is more addition than subtraction, then the fiber lengthens. If the subtraction rate is faster, then the fiber shortens.

Microtubule Assembly

Microtubules are assembled from subunits of a molecule called Tubulin. Tubulin comes in two forms" α-tubulin and β-tubulin. The α- and β-tubulin subunits fuse to form a tubulin heterodimer. The tubulin heterodimes then start lining up and adding to the "plus" end of the microtubule allowing it to grow in a spiral fashion. The result is a barber pole configuration. Viewed in cross section, it takes 13 of these heterodimers to complete a circuit of the microtubule. Viewed from the side, the heterodimers appear to be lined up vertically, these rows of heterodimers are referred to as protofilaments.

Centrosomes

Microtubules extend from a central point (yellow dot). This point is the centrosome. Typically, the minus end of all the microtubules originate from it. So that is where the tubulin heterodimers begvin building. The centrosome consist of a pair of centrioles that sit at right angles from each other. They are surrounded by an amorphous cloud of protein termed the pericentrosomal material. The initiation sites for the microtubules are in this material

How are the vesicles regulated between the rER and Golgi?

Movement of Transfer Vesicles between the rER and Golgi apparatus is regulated by the presence of two membrane bound proteins: Coat Protein Complex (COP) I and II. Transport Vesicles with COPII shuttle the rER products to the Golgi complex Return vesicles moving from the cis Golgi network to the rER are regulated by the presence of Coat Protein Complex I (COPI). These vesicles bring receptors and other useful molecules back to the rER. How to remember: COPII - two - go to the golgi COPI - one - "lonely" - Er return home

Ribosomes Build Proteins

Ribosomes are primarily made up of ribosomal ribonuceic acid (rRNA) and associated proteins. This rRNA is manufactured under the direction of DNA in the nucleus, and then exported to the cytoplasm. Active ribosomes in Eukaryotes are formed from two subunits: 60s and 40s particles that contain rRNA and a large number of associated proteins. When ribosomes attach to messenger ribonucleic acids (mRNA). This mRNA has been transcribed from deoxyribonucleic acid (DNA) in the nucleus and exported to the cytoplasm. Ribosomes provide the appropriate environment for the information in the mRNA to be translated into proteins (polypeptides) through the action of transfer ribonucleic acids (tRNA) attached to specific amino acids. Each 3- unit mRNA codon attracts a specific tRNA that adds a specific amino acid to the growing polypeptide.

Actin & Cell Movement

Several cell types in the body are capable of ameboid motion, most notably, macrophages. In this case, actin is added at the leading edge of the cell, and subtracted at the trailing edge, allowing the cell to glide along surfaces.

Actin & Myosin in Muscle

Some cell types are specialized to exert force on surrounding tissues. The most important of these are muscle cells (fibers). These cells are dominated by the presence of actin and myosin. In striated muscles, fibers made out of actin and myosin are arranged in very regular overlapping patterns that produce striations. Here this can be seen in light and electron micrographs taken of skeletal muscle fibers.

Cytosol visualization

The Cytosol is often just illustrated as space between the organelles (above), but it is actually jam-packed with free ribosomes, proteins and filaments, as indicated by this schematic (right). One wonders how there is room for the water that contains all these elements. The effect of the high concentrations is to increase the efficiency and activity of biochemical processes, because the constituents are so close to one another.

Golgi Apparatus

The Golgi apparatus receives proteins from the rER and modifies them before packaging them for shipment as vesicles. Ignore? --> The Golgi apparatus was first visualized by an Italian scientist who devised a silver stain to see these flattened cisterns. For this, Camilo Golgi received the Nobel Prize in 1906. We have sections using another of his silver stains for neurons in our slide set.

Centrioles

The centrioles are themselves composed primarily of tubulin. In this case the microtubules are formed as triplets that share some of their heterodimers.

cis-face of golgi apparatus

The cis-face is the forming face. Transfer vesicles from the rough endoplasmic reticulum (rER) form this side of the Golgi apparatus. (receiving side of the golgi) Usually, cis-face is convex.

Cytoskeleton

The cytoskeleton of the cell is made up of three elements: Microtubules - largest Intermediate Filaments Microfilaments (actin) - smallest

Peroxisomes

These membrane-bound organelles are used for degrading long chain fatty acids. They are smaller than lysosomes and secretory vacuoles. They are not visible at the LM level. Peroxisome enzymes degrade long chain fatty acids and form acetyl CoA and hydrogen peroxide as products. Peroxisome enzyme defects can produce lipid storage diseases. The acetyl CoA has multiple uses in the cell. Hydrogen peroxide is used in detoxification processes (e.g., for ethanol) and in some cells it is used to kill bacteria. However, levels of hydrogen peroxidase must be strictly controlled by catalase, as peroxide is highly toxic to cells.

Lysosomes at LM level

We generally can NOT see lysosomes at the light microscopic level in most cells. However, we can discern phagosomes in well fed macrophages. Chewed up bits of cells can sometimes be seen in macrophages at high magnification

Lysosomes

cell organelle filled with enzymes needed to break down certain materials in the cell Another product of the Golgi apparatus is the lysosomal vesicle (red arrows above). The lysosomes shown above reside in a macrophage, which uses them to digest the pathogens it ingests. ******Enzymes tagged with a manose-6- phosphate in the Golgi apparatus end up in lysosomes. ****Once they have fused with the targeted item, lysosomes are called phagosomes. ---->Note heterogeneity indicating two of these are actually phagosomes.

***How do you know the products from the golgi will end up in lysosomes?

the product vesicles will be tagged with mannose-6-phosphate (M6P)

Rough Endoplasmic Reticulum (rER)

the region of the endoplasmic reticulum that is studded with ribosomes and engages in protein modification. the ribosomes of rER can be seen as small, electron dense dots decorating the paired electron dense lines (railroad tracks) that indicate the membranes of the endoplasmic reticulum (ER). These membrane bound ribosomes on rER should be differentiated from free ribosomes found in the cytosol. Sets of free ribosomes can be organized into polyribosomes in the cytosol that are arranged into circular groups called rosettes.

Intermediate Filaments

very rigid and used for structure! Intermediate filaments (shown here in red) are like the girders of a cell. They give it it's shape and they keep it from being pulled apart. [Unlike the other fibers, they are not involved in motility, per se.] Intermediate filaments are attached to junctional specializations in the cell's membrane, shown above in green, allowing forces to be spread between cells and keeping tissues from being pulled apart.

Cilia and axonemes under LM and tEM

visible under the tEM and the LM

**explain how to know if a protein is made by rER

when the ribosome produce the polypeptide chain, there is a signal peptide sequence on the 5' end of the mRNA that signal will bind with the signal receptor protein (SRP) SRP will bring the newly synthesized polypeptide to the SRP receptor on the rER membrane SRP will free the polypeptide and thread it through the translocon on the rER membrane the translocon has a signal peptidase that will cut off the signal peptide sequence the polypeptide is made and moves into the cistern