Labs 2/3 – Cell Structure and Fates Cell ... - Indiana University · Cell Structure and Cell...

Cell Structure and Cell FatesLabs 2/3 – Cell Structure and Fates

IUSM – 2016

I. IntroductionII. Learning ObjectivesIII. KeywordsIV. Slides

A. Structures1. Plasma membrane2. Organelles

a. Nucleusb. Mitochondriac. Endoplasmic reticulum

i. Rough ER (rER)ii. Smooth ER (sER)

d. Golgi apparatuse. Lysosomes

3. InclusionsB. Cell Fates

1. Cell division (Mitosis)2. Cell death (Apoptosis)

V. Summary

Learning Objectives I – Organelles and Cytoskeleton

1. Understand how the cell membrane is “connected” to a system ofmembranous organelles in the cytoplasm and how synthesis ofmaterial in the cell relates to various modes of cell secretion.

2. Learn the appearance of common cytoplasmic organelles in light (LM)and electron microscope (EM) preparations.

3. Be able to correlate the two dimensional images of the organelles inthe LM and EM with their three dimensional structure.

4. Know the functions of the parts of these organelles and how theorganelles relate functionally to each other.

5. Understand the major features and differences of the components ofthe cytoskeleton.

Labs 2/3 – Cell Structure and FatesIUSM – 2016








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Learning Objectives II – Nucleus and Cell Cycle

1. Be able to induce the probable functional activity of a cell based onthe degree of development of its various cytoplasmic organelles andits nuclear morphology.

2. Learn the appearance of chromatin, nucleoli, and the nuclearenvelope in LM and EM preparations.

3. Visualize the three dimensional structure of the mitotic apparatusand understand the movements of chromosomes during mitosis.

4. Be able to identify the stages of cell division, the parts of the mitoticnucleus, and the mitotic apparatus.

5. Be able to recognize mitotic figures in routinely stained preparation.









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Keywords

AnaphaseApoptosisCentrioleCentrosomeChromatinCis-faceCisternaeCytoskeletonEuchromatinGlycocalyxGlycogen granulesGolgi apparatusHeterochromatinInclusionsIntermediate filamentsLipid dropletsLipofuscin granulesLysosomesMelanin granulesMetaphaseMicrofilamentsMicrotubules

MitochondriaMitosisMitotic figuresNuclear envelopeNuclear poresNucleolusNucleusPeroxisomesPlasma membranePolyribosomesPrimary lysosomeProphasePyknotic nucleusResidual bodiesRough endoplasmic reticulumSecondary lysosomeSecretory granulesSmooth endoplasmic reticulumStem cellsTelophaseTrans-faceVesicles









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Slide 151 (NW): Kidney, H&E

the plasma membrane is a phospholipid bilayer that bounds a cell; because of its high lipid content, it isusually difficult to see in most routine slide preparations; in this slide from the medulla of the kidney, the cellboundaries between cells of the collecting ducts are readily visible (in the renal lab, this will serve as a helpfulcharacteristic in distinguishing between the different “tubes” seen in the kidney)

plasma membraneis visible between cells of the collecting duct

why might the plasma membrane stain more readily for one cell type versus another?

plasma membranes are not visible between cells of the thick ascending limb









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https://vmicro.iusm.iu.edu/virtual_h_nw/nw_hist_1_151.html

Slide 29: Liver, H&E

nucleus of hepatocyte (liver cell)

nucleus of epithelial duct cell

nucleus of endothelial cell lining the lumen of blood vessel

nuclei of white blood cells; notice that the surrounding red blood cells (RBCs) lack nuclei

nucleus of connective tissue fibroblast

since the plasma membrane is generally not visible, the nucleus is usually the largest and most identifiablefeature of a cell; the shape of the nucleus often corresponds with the shape of the cell, and the size, location,and composition of the nucleus (e.g., presence of a nucleolus, amount of heterochromatin, etc.) are oftenhelpful in cell identification









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https://vmicro.iusm.iu.edu/virtual_h/msci_29_5.html


nucleus of a hepatocyte (liver cell) with heterochromatin, especially at the periphery of the nucleus lining the nuclear envelope, and giving it a basophilic appearance; unlike most cells in the body, it is not uncommon to see hepatocytes with more than one nucleus

nucleus = double membrane enclosing chromatin (proteins + DNA [acid]) = binds hematoxylin [base]









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Slide 44: Pancreas, H&ESlide Overview

most of the “spaces” are adipocytes (fat cells)

lymph node

light-staining regions are pancreatic islets which are responsible for the endocrine functions of the pancreas (e.g., insulin production)

peripheral nerve

large, blood-filled vein; note that blood is usually seen “pooled” in veins, not in arteries









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https://vmicro.iusm.iu.edu/virtual_h/044_bl_5.html

Slide 44: Pancreas, H&ELabs 2/3 – Cell Structure and Fates

IUSM – 2016








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nuclei of pancreatic secretory exocrine cells; the nuclei are located in the basal (bottom) aspect of the cells, not the center

why is the nucleus located basally instead of apically?

small, eosinophilic secretory granules located in the apical aspect (top) of the cell

the majority of the parenchyma(functional part of an organ) of the pancreas is composed of secretory exocrine cells that produce digestive enzymes


Slide 71: Cerebellum, Cresyl Violet

brainstemlook in the brainstem to find examples of motor neurons with large nuclei and prominent nucleoli

cerebellumlook in there cerebellum to find several different types of neurons with various sized nuclei

Slide Overview









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Slide 71: Cerebellum, Cresyl VioletLabs 2/3 – Cell Structure and Fates

IUSM – 2016








V. Summary

nucleus of large Purkinje cell with a prominent dark-staining nucleolus in the center

nuclei of small granule cells(the most numerous type of neuron in the brain)

nucleus of a motor neuron with a small, dark central nucleolus; very little cytoplasm is seen surrounding the nucleus

disgruntled histology student?(artifact)

nucleus of a neuroglial cell


Slide 9: Spinal Cord, H&E

nucleus of a motor neuron; notice the large basophilic nucleolus and the many small patches of heterochromatin(condensed chromatin)

lots of basophilic rER can be seen surrounding the nucleus, outside the nuclear envelope, in the lighter-staining cytoplasm









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Slide 101: Stomach, H&E

mitochondria are the “powerhouses” of the cell; they are surrounded by double membranes, with the innermembrane folded to form cristae; mitochondria are usually easily recognizable in electron micrographs butare not seen in routine light microscopy; however, mitochondria are acidophilic so they can influence thestaining pattern of the cytoplasm, as seen in the highly-eosinophilic cytoplasm of gastric parietal cells

parietal cells in the stomach have a characteristic “fried egg” appearance with a central nucleus and intensely-staining eosinophilic cytoplasm from the large number of mitochondriapresent









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Slide 154: Pancreas, H&E

dark, basophilic patches near the nucleus in the cytoplasm are the rough endoplasmic reticulum(rER)

rER = membranous sacs + ribosomes (rRNA) [acid] = binds hematoxylin [base]

eosinophilic secretory granules

the endoplasmic reticulum is network of membranous sacs (cisternae) which are continuous with the nuclearenvelope; the membranes themselves do not stain in routine preparations, but when polyribosomes are bound(making it “rough” endoplasmic reticulum) they bind basic stains and create a basophilic region of cytoplasm









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Slide 71: Cerebellum, Cresyl Violet

in neurons the rER is especially prominent and referred to as Nissl (chromatophilic) substance

nucleus

nucleolus









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Slide 75: Adrenal Gland, H&ESlide Overview

medullalook in this lighter-stained layer (the zona fasciculata) to find steroid-producing cells with abundant smooth endoplasmic reticulum (sER)

the cortex (outer portion) of the adrenal gland is a major site of hormone production in the body; its threedistinctive layers, noted by their differences in staining, are each primarily responsible for a specific class ofhormone, for instance, the zona fasciculata is the site of glucocorticoid (e.g., cortisol) production









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Slide 75: Adrenal Gland, H&E

as the sER lacks associated ribosomes (with rRNA) it stains poorly and gives a speckled (“moth-eaten”) appearance to the cytoplasm

sER = membranous sacs + no ribosomes = no H&E stain binding









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like the cells of the adrenal cortex, hepatocytes (liver cells) are also rich in sER - for various functions - so have regions of cytoplasm that appear empty or unstained

hepatocytes are abundant in most organelles: they have lots of smooth and rough endoplasmic reticulum, lots of mitochondria, lots of Golgi, lots of lysosomes, lots of inclusions such as glycogen granules, and they may even be multi-nucleated









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Slide 42: Lymph Node, H&E

Slide 10: Blood Smear

continuing the trend of difficult to see structures, the Golgi apparatus is group of flattened membranous discscalled cisternae formed from vesicles from the endoplasmic reticulum; as such, it does not readily stain in mostroutine light microscopy preparations; however, as the Golgi serves an important role in protein secretion, it canbe abundant enough in certain cells to produce a non-staining region typically located near the nucleus; this isoften referred to as a “negative” or “ghost” of the Golgi and can be see directly at the arrow tips in the plasma cell(slide 42) and lymphocyte (slide 10) above









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https://vmicro.iusm.iu.edu/virtual_h/10_2_bl_5.html

Slide 135: Sympathetic Ganglion, H&E

in these neurons of a sympathetic ganglion (a collection of neuron cell bodies outside the CNS), the brown/rust colored pigment in the cytoplasm are residual bodies of lipofuscin (lip-oh-fuss-sin)granules, a “wear-and-tear” pigment, in the remains of secondary lysosomes; these are generally found in long-lived cells









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lysosomes are difficult to see in LM; however, in certain cell types, they might be visible due to the presence of indigestible residues










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inclusions are cytoplasmic accumulations not bound by a membrane; they include lipid droplets which aretypically removed from cells during slide preparation so generally appear as empty or unstained vacuoleswithin a cell, as seen at the arrow tips in the secretory cells of the mammary gland (slide 108); melaningranules are dark brown/black pigmented granules found several places within the body including the skin(slide 36), as indicated by the arrow

Slide 108: Mammary Gland, H&E

Slide 36: Thin Skin, H&E











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glycogen granules are another type of inclusion; they can be abundant in hepatocytes but can be difficultto clearly identify in standard H&E preparations; the image on the left shows a PAS-stained liver, with thearrows indicating glycogen granules; the image on the right is the liver after treatment with amylase(breaks down glycogen), showing the depletion of glycogen, as occurs in fasting or exhaustive exercise

Image source: Digitized Atlas of Mouse Liver Lesions, NIEHS









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Slide 93: Onion Root Tip, H&ESlide Overview

look in the darker regions of the tips (apical meristem) where mitotic cells can be seen more clearly

Why onion roots?

1. Cells are rapidly-dividing

2. Large, visible chromosomes

• Genome is ~6x larger than humans

• 2n = 16 (vs. 46), so individual chromosomes are larger

3. Visible cell walls and ability for cells to be flattened for slide preparation










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Slide 93: Onion Root Tip, H&E

(2)metaphase

(4)telophase

(3)anaphase

(1)prophase

prophase vs. interphase: cells in interphase have less-condensed chromatin (i.e., chromosomes not visible)and generally have a more-visible nucleolus (maybe more than one) which disappears during prophase










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stem cells in the intestinal crypts undergo asymmetric division, producing one stem cell and a non-stem cell progeny that then begins to differentiate and migrate up to the tips of the villi where they are sloughed off in the lumen; the entire process usually takes less than one week

Slide 37: Ileum, H&ESlide Overview

villi are folds of tissue of the intestinal wall that serve to increase surface area for absorption

(lumen)

look in the intestinal crypts/glands for mitotic figures










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Slide 37: Ileum, H&E

the stages of mitosis are difficult to see on routine slides of animal tissue, so cells undergoing mitosis arereferred to generally as mitotic figures which are distinguished by abundant heterochromatin (chromosomes)and lack of a defined nucleus; they can be found within certain tissues of adult organs by looking for dense,irregular shapes instead of a normal nucleus

mitotic figures in intestinal crypt/gland

why might stem cells be restricted to only certain areas of an organ, such as in the intestinal crypts?

eosinophilic secretory granules










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Slide 136: Tonsil, H&E

look in the lymphoid follicles

(collections of lymphocytes)for mitotic figures










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Slide 136: Tonsil, H&E

mitotic figures in lymphoid follicle










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Slide 80: Endometrium, H&E

look in theendometrial glands for mitotic figures; the endometrium is the inner lining of the uterus andexperiences rapid proliferation each month as part of the normal menstrual cycle; the thickness of thelayer greatly increases as does the prevalence of the endometrial glands, making them a good area tolook for cells undergoing mitosis

http://medsci.indiana.edu/histo/virtual/080_bl_5.html









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Slide 80: Endometrium, H&E

mitotic figures in an endometrial gland

http://medsci.indiana.edu/histo/virtual/080_bl_5.html









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Slide 4: Duodenum, PAS

cells that began in the intestinal crypts (i.e., mitotic figures) migrate up the length of the villi toward theintestinal lumen where they will undergo apoptosis (die) and be shed into the lumen as waste

villi line the lumen

crypts

crypts










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Slide 4: Duodenum, PAS

cells undergoing apoptosis

to identify cells that are undergoing apoptosis (a form of programmed cell death), look for nuclei that areout of line with neighboring cells due to loss of cell-to-cell junctions, pyknotic nuclei (dense mass ofchromatin with no indication of chromosomes), and condensed eosinophilic cytoplasm










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Slide 156: Skin, UV Damaged, H&Eapoptotic cell with pyknotic nucleusand eosinophilic cytoplasm










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Common Confusion:Mitotic Figure vs. Apoptosis

Slide 136, Tonsil, H&E Slide 37, Ileum, H&E

Slide 156, Skin, H&E Slide 4, Duodenum, PAS

Mitotic cells: cells undergoing cell division(mitosis); most often seen in cells of actively-dividing tissues (may be restricted to specificlayers) including skin, bone marrow, gonads,and the gastrointestinal tract

Look for: (1) lack of nuclear membrane anddefined nucleolus; (2) dense, irregular shapeswhere the nucleus would normally be located;may also appear as one or two dark, straightlines in the nucleus (a straight or bar-shaped‘nucleus’ represents mitosis and not apoptosis)

Apoptosis: a type of physiologic programmedcell death which occurs 20x faster thanmitosis, so is less likely to be seen in routinepreparations; also, likely to occur in differentregions of tissue from mitosis

Look for: (1) pyknotic nuclei and cell nucleiout of line from adjacent cells; (2) condensed,eosinophilic cytoplasm; (3) formation ofapoptotic bodies









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Summary

1. Most organelles and cytoplasmic elements are difficult to distinguish in standard LM and are bestviewed in EM micrographs or through specialized staining methods (e.g., immunocytochemistry).

2. The cell nucleus is generally the largest and most easily seen organelle in the cell due to itsabundant basophilic chromatin; nucleoli (“little nuclei”) are intensely-staining basophilic regions(due to rRNA) normally located in the center of nuclei where ribosomal subunits are assembled.

3. The endoplasmic reticulum network is continuous with the nuclear membrane: rER is viewablein LM due to the basophilic staining properties of the rRNA of the attached polyribosomes; thepresence of sER can be inferred from the absence of stained areas in the cytoplasm, especially incells which secrete complex lipids (e.g., steroid hormone producing cells).

4. Other organelles include the Golgi apparatus, generally seen as a non-staining “ghost” regionadjacent to the nucleus, and lysosomes, which can at times be visualized by the presence ofpigmented digestion residues (e.g., lipofuscin).

5. The stages of mitosis are difficult to see on slides of animal tissue, so cells undergoing mitosis arereferred to generally as mitotic figures which are distinguished by abundant heterochromatin(chromosomes) and lack of a defined nucleus.

6. Cells underjoining apoptosis are distinguished by pyknotic nuclei and condensed, eosinophiliccytoplasm.

Characteristic Appearance

Structure Electron Microscopy Light Microscopy (H&E stain)

Nucleus

Mitochondria

rER

sER

Golgi

Lysosome

Peroxisome

Lipid droplet

Melanin granules

Mitotic figure

Apoptotic cell

Appearance of Cellular Structures in Electron and Light MicroscopyLabs 2/3 – Cell Structure and FatesIUSM – 2016








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