Intermediá rne Filamenty

Katarína GaplovskáKatedra genetiky

25 nm

Microtubules

5-9 nm

Microfilaments

Intermediate Filaments (IF)

10 nm

Actin Focal adhesions

MicrotubulesGolgi apparatus

CytokeratinsDesmosomes

transmit mechanicalforces between adjacent cells

Lamin A

Two types of IF in metazoan cells

Vimentin

J Clin Invest. 2009 July 1; 119(7): 1772– 1783.

DNA

Two distinct cytoplasmic IFs in one cell

Human alveolar carcinoma cells

J Clin Invest. 2009 July 1; 119(7): 1763– 1771

General features of IF• encoded in human genome by 70 different genes

• conserved α-helical rod domain, N- and C-terminal domains of different sizes

• lamins generally expressed, cytoplasmic IF – tissue specificexpression, each class of IF unique properties

• IF evolution – Lamin gene duplication -> deletion of NLS and CAAX box = cytoplasmic intermediate filaments

Major classes of IF in Mammals

IF structure

IF structure

• common core structure

• contain above-averrage hydrophobic residues and sequences

• alpha-helical conformation with heptad repeats

• 2 monomers form Coiled-coil structure

Coiled-coil structure is typical for IF

• 3,6 amino acid per α-helix turn• position a and d of heptad repeat

= hydrophobic amino acidsLeu Val Met

Assembly of IF

J Biol Chem. 2007 Jun 22;282(25):18563-72.

IF assembly in vitro

ULF = UnitLengthFilament

soluble tetrameric complexes of wild-type desmin

IF assembly in vivo

End to and assembly

Posttranslational modification of IF

Phosphorylation

• lamin & vimentin = disassembly nuclear Lamina and vimentin filaments

• keratins et al. = partial disassembly

• neurofilaments = heavily phosphorylated C-termstabilization

Disulfide and amide bonds formation

• keratins = „fiberglass-like“ material

IF dynamicsIntermediate filaments: • have long half-lives and biochemicaly are stable

• disassembly and reassembly during cell spreading, wound healing and cell division etc.

vimentin subunit exchange –FRAP experiment

Mechanical properties of microfilaments,microtubules and intermediate filaments

J. Cell Biol., 1991 113:155– 160

Atomic Force Microscopy

Soft - flexible –nearly unbreakable

Herrmann et al., Nat Rev Mol CellBiol. 2007 Jul;8(7):562-73

IF-pathies

Keratins• major proteins in skin, epithelial cells, hair and nails• most diverse IF family (>50)• form heterodimers = acidic + basic keratin• diagnostic tool for carcinomas• disulfide bridges

Basal Lamina

Epidermis

>10%

>70%

Defects in Keratins cause disease

Epidermolysis bullosa simplex

• rare genetic disorder with bullous lesions

• skin is fragile and blisters

• K5 & K14 impaired

type I and II intermediate filament

• basal keratinocytes become fragile

Epidermolysis bullosa simplex

Vimentin

lymphocyte

vimentin IF network in cage configuration = resistance to

haemodynamic and mechanic stresses

1. chemokine induced chemotaxis

2. Site-specific phosphorylation

3. perinuclear relocalization

4. extravasationinflamation

Desmin• connects contractile apparatus and structural elements of the cell

• in structures surrounding Z- disk(skeletal muscle)ordesmosomes (cardiac muscle)

Desmin

Normal myocyte

Goldfarb & Dalakas, J Clin Invest. 2009;119(7):1806– 1813

Desminopathy

mutant advanced stage

myofibrillar myopathy, cardiomyopathy etc. => aggregation ofmisfolded protein

Glial fibrillary acidic protein (GFAP)

• in astroglial cells • support and nurishment of neurons in brain• probably involved in controlling the shape, movement,

and function of astroglial cells

GFAP - astrocyte

Neuronal tubulin - neurons

Alexandre disease

• neurodegenerative disorder

• loss of myelin (degeneration of white matter in brain)

• enlarged brain and head size, etc.

• mutant protein acts in gain-of-function fashion

wild type astrocytes

Mignot et al., Exp Cell Res. 2007, 313(13):2766-79. video

Motor neuron

Axon

NF-H MT

NF-M

NF-HC- terminal tail domains = IF spacing

Subcortical actin

Neurofilaments

Lamins

• Lamin A/C – in differentiated cells

• Lamin B1 and Lamin B2- essential

• Form nuclear lamina

Lamins

Role in:

• controling nuclear shape

& size

• DNA replication

• DNA transcription

• Cell signaling

• Structural, functional and

epigenetic organization of

chromatin etc.

Lamins & cell division

Lamin B & Mitotic Spindle Matrix

Lamin Bessential for spindle matrix formation

Spindle matrixpromotes microtubule assembly and

organization in mitosis

Tsai at al., Science. 2006 Mar 31;311(5769):1887-93

Laminopathies

Hutchinson-Gilford Progeria syndrom(HGPS)

• Lamin A mutated• premature aging disorder• incidence 1 per 4– 8 million live births • average life expectancy for a patient is 13 years (7 – 27y)• 97% caucasian origin

Coutinho et al., Immunity & Ageing 2009, 6:4

Lamin A

Lamin A-Progerin

Mutant Lamin A alters mitosis & cell cycle progression

Dechat et al., Proc Natl Acad Sci U S A. 2007 Mar 20;104(12):4955-60

Plectin cross-links IF to Microtubules