Cell (2)黃敏銓

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解剖學暨細胞生物學研究所

mchuang@ntu.edu.tw

Lysosome:The stomach of the cell

• Single membrane-bounded

• Containing over 60 acid hydrolases(optimal pH 5.0): marker enzyme/acid phosphatase

• Pinocytosis, phagocytosis, receptor-mediated endocytosis, autophagy

• Lysosomal storage diseases: about 50 inherited metabolic disorders, deficiency in galactosidase (neurons)

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Classification of lysosomes

• Primary lysosomes

• Secondary lysosomes: primary lysosomes fused with phagosomes, autophagosomes

• Residual bodies

• Lipofuscin pigments

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Lysosome biogenesis

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Pathways deliver materials for lysosomaldegradation:

Extracellular (large): phagocytosis

Extracellular (small): pinocytosis, receptor-mediated endocytosis

Intracellular: autophagy

Primary lysosome

Residual bodiesphagosome

Autophagicvacuole

lipofuscin pigment6

Primary lysosome

Hepatocyte

Histochemical staining of lysosomes for acid phosphatase 7

Lysosomes (Lipofuscin)

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Lysosomes9

Secondary lysosome

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Primary lysosome

Phagosome and lysosome

Bacteria in neutrophils

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Ps: phagosome

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Endosome maturation

The EMBO Journal (2011) 30, 3481–3500

Early endosome

Late endosome

Intraluminal vesicle (ILV)

Multivesicular body (MVB) / Late endosome

Lysosome

TGN

Multivesicularbody (MVB)

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Peroxisomes (microbodies)• 0.5 µm in diameter

• Single membrane-bounded

• Containing oxidative enzymes, paricularlycatalase and other peroxidases, urateoxidase (crystalline core, crystalloid inclusion, nucleoid, not in humans): lipid metabolism & detoxification

• Toxic effects of H2O2 produced by metabolism (AA, FA) are prevented by peroxisomes

• Zellweger syndrome: failure of transporting peroxisomal proteins into peroxisomes, early death

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Peroxisomes with a crystalloid inclusion (nucleoid)16

Peroxidase histochemical staining: only Peroxisomes are positive 17

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Autophagosome

Mitochondria• Generating ATP

• Localized at the sites where energy is needed (intermyofibrillar space, basal infoldings)

• Evolved from aerobic bacteria

• Mitochondrial proteins: encoded by mitochondrial and nuclear DNA

• Mitochondria decide if the cell lives or dies

– Cytochrome c : initiating apoptosis signaling

• Eosinophilic (because of membranes)

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1. Outer membrane: permeable to <5000 Da.

2. Inner membrane: cristae (folds) (elementary particles/oxidative phosphorylation sites)

3. Matrix: Krebs cycle enzymes, matrix granules (Ca2+

storage), mitochondrial DNA, tRNAs, ribosomes 20

Iron hematoxylin (mitochondria)

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Salivary gland duct

Striations

capillary

M

Basal membraneinfolding

M

M

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Basal infolding (striation)

23Kidney tubule

Succinate dehydrogenase stain

Red muscle fiber

White muscle fiber

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rER

Matrix granule

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Cytochrome oxidase histochemical stain (EM)

mitochondria 26

Intermembrane space

Lamellated cristae

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M

Lipid

myofibrils

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CytoskeletonComponents: microtubules, intermediate

filaments, actin filaments

• Microtubules (25 nm) :

centrioles, spindle fibers, cilia, flagella

• Filaments:

intermediate filaments (10 nm)

actin filaments (7 nm)

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EM of microtubules (25 nm in diameter)

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• 5 nm thick wall formed by 13protofilaments consisted of dimeric tubulin molecules (and -tubulin), MAPs (, kinesin, dynein etc.)

•Dynamic instability: Dimersare added or removed dynamically

33(- end)

(+ end)

Molecular motors1. Dynein: moving organelles from the cell

periphery toward the MTOC (minus “-” end) 2. Kinesin: moving organelles from the cell

center toward the cell periphery (plus “+”end)

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Cell periphery

Cell center

Microtubules growing from -tubulin rings (nucleation site) within the MTOC (Centrosome, …)

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(not in humans)

Centrosome: centrioles + pericentriolar material

metaphase

anaphase

telophase

Mitotic spindles

Green: spindle microtubulesBlue: chromosomes 36

Location of MTOCis visualized by centrin staining(red). Microtubules (green). Nucleus(blue)

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Microtubules radiate from MTOC (Centrosome)

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Centrioles: green Kinesin: redChromosomes: blue Kinetochore: white

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Centriole

• 0.2 m x 0.5 m

• 9 microtubule triplets

• Located in centrosome(centrioles+pericentriolarmaterial)

• Forming basal bodies for ciliogenesis

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EM of paired centrioles

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Centrioles duplicate during mitosis:Parent and daughtercentrioles in a fibroblast

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43University of Waikato. Tony Poole

Cilia: Axoneme: core complex composed of 9 microtubule doublets + 2 central microtubules (9x2 + 2)(motile cilia & flagella)

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PseudostratifiedColumnar epithelium

Basal body

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EM: Cross section of cilia

Microvilli

Cilia

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Basal bodies and cilia (longitudinally sectioned)

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G-actin F-actin2 F-actin actin filaments

1. Cytochalasin B or D: binding to the plus end of actin filament, inhibiting phagocytosis, cell division, cell migration

2. Phalloidin: binds to F-actin, causing cell death

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ActinPolymerization of actin filaments

Nucleus: DAPIMitochondria: MitoTracker red actin bundles (stress fibers): stained by NDB phallacidin 49

Thin filaments in a cardiac myocyte

Red: tropomudulinGreen: actin

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Thin filaments in cardiac cells

IF

actin

microvilli

Terminal web

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Intermediate filaments

Rope-like

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Silver-impregnation: (neurofilaments)brown cytoskeleton in neurons 53

Intermediate filaments: non-polar, highly variable, rope-like

Class 1 and 2: keratinsClass 3: vimentin and vimentin-like

(desmin, GFAP, peripherin)Class 4: neurofilamentsClass 5: laminsClass 6: beaded filaments (lens specific)

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Cytoplasmic inclusions

• Lipid droplets: no limiting membrane, lipid contents are stained by osmium tetroxide and Sudan black

• Glycogen: 25 - 30 nm particles

• Pigment granules: melanin granules, lipofuscin, hemosiderin granules

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Glycogen (red): PAS-hematoxylin stained hepatocytes

Glycogen

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Glycogen particles: 25-30 nm, electron-density higher than ribosomes (15-20 nm), β-particle

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Brown adipose tissue

HE Osmium

Lipid droplets

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Mitochondria 0.2-2 x 2-7 m Double membranes, cristae

Lysosome 0.2 - 0.5 m Single membrane, oval to polymorphic, hydrolases(acid phosphatase)

Peroxisome 0.2 - 0.5 m Single membrane, spherical, catalase and peroxidase, nucleoid

Cilia (flagella) 0.2 x 10 m membrane bounded, cylinder, 9 doublets (9x2)+ 2 siglets

centriole (Basal body)

0.2 x 0.5 m No membrane bounded, paired cylinders, 9 triplets (9x3)

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