Non-membranous cell organelles.Cytoskeleton – structure, function

and tissue specificity. Specialization of the cell surface.

Intercellular junctions.Biological motors – molecular principles

Institute of Histology and EmbryologyAuthor: Prof. MUDr. Jindřich Martínek, DrSc.

Subject: General Histology and general embryology Code: 82241Date: 2013, October, 10th and 12th

NON-MEMBRANOUS CELL ORGANELLS

• NUCLEOLUS• RIBOSOME• CENTRIOLE• CYTOSKELETON

thin filaments - actin

intermediate filament

lamins (A, B, C)

cytokeratins (1 – 20)

desmin, vimentin

nestin

microtubules

Becker: The world of the cell, 1986

NON-MEMBRANOUS CELL ORGANELLES

• NUCLEOLUS – localization in the karyoplasm – nucleolar organizing centers (NORs) - two components (amorphous and granulous) – site of transcription of r-RNAs (ribosomal) – compact, network and ring-form type

• CENTRIOLE – specific organization of microtubeles (9 triplets) – MTOC – microtubular organizing center function, simple and multiple centriolar replication. Role in formation of mitotic and meiotic spindle and basal bodies of cilia and flagella

• CYTOSKELETON – complex of thin (5-7 nm) and intermediate (10-15 nm) filaments and microtubules (24 nm) – actin (thin) filaments with myosin and tubulins (of microtubules) with dynein and kinesin represent molecular motors for intracellular traffic – intermediate filaments are nonpolar – nestin, cytokeratins, vimentin, desmin – tissue specific – cells of epithelial origin (cytokeratin), mesenchymal origin (desmin), muscle tissue (vimentin)

• RIBOSOMES – (15-30 nm) composed of two subunits (small and large) – play a role in translation – protein synthesis – as free polysomes and membrane bounded (GER)

PF

PG

FC

PNC

PNC

Types: compact – initial transcription of rRNA at the single NORs; network – massive transcription in gradually confluent NORs ring-form – decreasing transcription at the single NOR

NUCLELUS – non-membranous cell organelle derived from activated parts of some chromosomes – nucleolar organizing regions (NORs) or nucleolar organizing centers (NOCs)

FC = fibrilar center – NOCPF = pars fibrosa – accumulated transcriptPG = pars granulosa – ribosomal assemblyPNC = perinucleolar chromatin

VISUALISATION OF NUCLEOLAR ORGANISERS USING IMMUNOHISTOCHEMICAL PROBES

TYPES OF NUCLEOLI:a) compact – currently activated nucleolar

organiser b) with nucleolonema – massive

formation of ribonucleoproteins

around NORs - typical network appearancec) ring form – decreased of RNA transcription in

the inactivated nucleolar organiser

1

1

1 = perinucleolar chromatin 2 = nucleolar organiser3 = nucleolonemata 4 = nuclear channel system5 = lamelae annulatae 6 = lysosomes

2

2

3

3

4

56

RIBOSOMES (15 – 30 nm)

free polysomesGERPROTEIN SYNTHESIS

GER

NUCLEUS

NUCLEARPORES

GER

RIBOSOME – TRANSLATION MECHANISM – POLYSOMES

Start codon (AUG)

Currently translating codon (arbitrary)

Scheme of the protein synthesis on freepolysomes (Junqueira´s Basic Histology, Mesher, 2010)

ribosome

mRNA

free proteinin the cytoplasm

Electron micrograph: Histology, Ross, Pawlina 2010Arrows: free polysomesRough (Granular) Endoplasmic Reticulum rER (GER)

GER

GERGER

IMMUNOHISTOCHEMICAL DETECTION OF SOME CYTOSKELATAL COMPONENTSMicrotubules (red), actin filaments (green)– fluorescence microscope

CHARACTERISTICS OF CYTOSKELETAL COMPONENETS

Diameter: 6 – 8 nm 10 – 12 nm 24 nm

Composition: Polymer of G-actin Various proteins Dimers of α- and ß-tubulin

Structure: Double-stranded F-actin helix Ropelike fiber Hollow non-branched cylinder Thin flexible filament Strong, stable structure Exhibit dynamic instability Readily dissociate and reassamble Enzymeactivity: ATP hydrolitic activity None GTP hydrolytic activity

ATP-dependent polymeration GTP-dependent polymerationLocation and functionin the cell: Terminal web Extend across cytoplasm Core of cilia and flagellum Zonula adherens connecting desmosomes Centriole Core of microvilli and hemidesmosomes Mitotic spindle Contractile ring in the Nuclear lamina of nucleus Provide network “railroad dividing cell Support of cell processes tracks“ for movement of Contractile elements Provide mechanical strenght organelles within cell of muscles and resistence to shearing Movement cilia and forces chromosomes (during cell division

Ross, Pawlina: Histology, 2006

Actin filaments Intermediate filaments Microtubules

ACTINthin filaments (5 – 7 nm)G and F actin – ATP dependent

(poly- and depolamerization)

MOLECULAR MOTOR COMPONENTtogether with myosin

Smooth muscle cells

Hearth muscle cells - cardiomyocytes

FUNCTION OF MICROFILAMENTSAND MICROTUBULES IN REGULATIONSAND INTRACELLULAR TRAFIC

THE MOLECULAR MOTOR PROTEINS WORKING WITH MICROFILAMENTS (ACTIN)

Unipolarly organized myosin(myosin monomers – myosin I) –one way movement of cargo

Bipolar organization of myosin(myosin monomers – myosin II) –ontractile activity with oppositedirection movement of actinfilaments – muscle tissue

Unipolarly working myosin (I) –attached to the cytoplasmicmembrane – formation of pseudo-podia movement of cell

THE MOLECULAR MOTOR PROTEINS WORKING WITH TUBULINS OF MICROTUBULES

Scheme: Histology, Ross, Pawlina, 2010

Kinesins move along the MT tothe plus end and can transport cargo (organelles) from the cytocentrum toward the cell peripheryDyneins move along the MT to the minus end, transport cargo (endocytotic vesicles) from the cell periphery toward the MTOC.

INTERMEDIATE FILAMENTS – non-polar lamins A, B, C – nuclear lamina

cytokeratins – cells of germ layer origin

desmin – cells of mesenchymal origin

vimentin – cells of mesenchymal origin neurofilaments – nerve cells

GFAP – glial fibrillary associated protein (acidic) nestin – during cell development

CYTOKERATIN 18 – fission products – APOPTOTIC MARKER

MICROTUBULES (25 nm) tubulin α, β, γ (GTP – dependent polymerization)MOLECULAR MOTOR COMPONENT – together with dynein (+ -) and kinesin (- +)

CENTRIOLAR PAIR - CENTROSOMECILIUM – KINOCILIUM

Ross, Pawlina: Histology, 2006

CENTRIOLES AND CENTRIOLAR REPLICATION

SIMPLE REPLICATION – begins fromexisting centriolar pair

MULTIPLE REPLICATION – starts as synthesis of precursor material (tubulins)and continues as induced assembly of nine procentriolar triplets of microtubules – typical for cells with kinociliary apparatus at the apical surface

Centriolar pair = diplosome – centrosomeCentriole: Procentriole: = 200 nm = 200 nm L = 400 nm L = 200 – 400 nmCentriolar DNA – MTOC – centriolar precursors

Mucus layer

ZONULA OCCLUDENS – freeze fracturing technique

ZONULA OCCLUDENS – tight junctions – missing intercelluar space

RidgesGrooves

Ross, Pawlina: Histology, 2006

Ciliarynecklaces

Golgi complex

Zonula occludens

Desmosome – macula adherens

Cytokeratin – intermediate filaments

Zonula adherens

ZONULA ADHERENS – terminal web – transverally arranged actin filaments inserting via α-actinin and vinculin and catenin into E-cadherin moleculs

GAP JUNCTIONS – NEXUSSES –communicative cell to cell interconnectionsconnexon – canal formationconnexins – membrane proteins

MICROVILLI = 0.1 mlenght = 1 – 5 m – core – actin filamentsRegularly arranged and numerous microvillirepresent specific surface specialization of anabsorptive epithelium – brush border –at the apical pole of cells.Stereocilium – long and branched microvillus

Ross, Pawlina: Histology, 2006

Microvilli at the cell suface and intercellular interconnectionZO = zonula occludens; ZA = zonula adherens; D = desmosome

zoZA

D

SOLITARY (single or individual) CILIUM

Basal body

Axonemal complex

Ciliary sheatPinocytotic vesicle

= 0.25 mL = 3 – 5 (7) m (50 for tail of spermatozoon) Consist of basal body (9 triplets – MTOC) and axonemal complex (9 doubletsand 1 central pair of microtubules).Basal body develops from one ofcentrilar pair and therefore it is lo-cated often in the CYTOCENTRUM.

Ross, Pawlina: Histology, 2006

KINOCILIARY APPARATUS

Oviduct – simple columnar ciliated epithelium

Cilia and microvilliat the cross section

INTERDIGITATIONS

CILIA, MICROVILLI, JUNCTIONAL COMPLEXESAND TERMINAL WEB

IMMUNOFLUORESCENT DETECTIONOF PANCADHERIN IN SEMINIFEROUS TUBULES OF TESTIS (Zonulae adherentes)BETWEEN SERTOLI CELLS PROCESSES

BASOLATERAL LABYRINT

Typical for absorptive type ofepithelial cells as in theproximal tubules (kidney)or in lining of so calledstriated ducts in somesalivary glands.Specific for an intesiveIon transport (energyneeded) by e.g. Na+, K+ ATP-ase asan integral protein of the plasma membrane

Stevens, Lowe: Histology, 1993

Stevens, Lowe: Histology, 1993Ross, Pawlina: Histology, 2006