Research

Microtubule reorganisation is critical for differentiation, tissue formation and function and this is particularly evident during apico-basal polarisation of epithelial cells such as those of the kidney, intestine and inner ear.

Relatively flat undifferentiated cells have a classic radial array of microtubules emanating from the centrosome whereas many differentiated epithelial cells contain apico-basal microtubule bundles that are often non-centrosomal.  - Diagram by Selina Catto

Relatively flat undifferentiated cells have a classic radial array of microtubules emanating from the centrosome whereas many differentiated epithelial cells contain apico-basal microtubule bundles that are often non-centrosomal. – Diagram by Selina Catto

EB2 is required for microtubule reorganisation and epithelial differentiation

Microtubule end-binding (EB) proteins influence microtubule dynamic instability, a process essential for microtubule reorganisation. We have recently establish a role for EB2 in microtubule reorganisation during apico-basal epithelial differentiation.

Goldspink et al 2013. J Cell Sci. 126: 4000-4014

ARPE-19 cell showing EB2 (green) at Mt (red) plus-ends and along the lattice. Scale bar 10μm - Image by Deborah Goldspink

ARPE-19 cell showing EB2 (green) at Mt (red) plus-ends and along the lattice. Scale bar 10μm – Image by Deborah Goldspink

EB2 is required for initial microtubule reorganisation but its down-regulation leads to microtubule stability, EB1 and ACF7 lattice association, co-alignment with actin filaments and bundle formation.

Goldspink et al 2013. J Cell Sci. 126: 4000-4014

EB2 siRNA depleted epithelial cell with EB1 (blue) along the microtubule (red) lattice. Scale bar = 10μm. Image by Deborah Goldspink.

EB2 siRNA depleted epithelial cell with EB1 (blue) along the microtubule (red) lattice. Scale bar = 10μm. Image by Deborah Goldspink.

Inner ear and intestinal crypt epithelial tissues show a direct correlation between low level of EB2 expression and the presence of apico-basal microtubule bundles, which are absent where EB2 is elevated.

Inner ear with high EB2 (green) expression in hair cells and low in supporting cells. Scale bar 5μm. Image by D. Goldspink

Inner ear with high EB2 (green) expression in hair cells and low in supporting cells. Scale bar 5μm. Image by D. Goldspink

EB2 (red) is highly expressed in basal intestinal crypt cells where unbundled curvy Mts (blue) predominate whereas in differentiating cells EB2 expression is low and distinct Mt bundles are evident. Scale bar 5μm. Image by J. Gadsby

EB2 (red) is highly expressed in basal intestinal crypt cells where unbundled curvy Mts (blue) predominate whereas in differentiating cells EB2 expression is low and distinct Mt bundles are evident. Scale bar 5μm. Image by J. Gadsby

The level of EB2 expression influences microtubule stability and dynamics

EB2 siRNA depletion or its down-regulation during epithelial differentiation increases microtubule stability and decreases microtubule dynamics.

Detyrosinated tubulin (green) is prominent in microtubule bundles in EB2 siRNA depleted cells. Image by Jonathan Gadsby.

Detyrosinated tubulin (green) is prominent in microtubule bundles in EB2 siRNA depleted cells. Image by Jonathan Gadsby.

EB2 depleted cells contain more cold-resistant microtubules than control cells. Image by Jennifer Keynton.

EB2 depleted cells contain more cold-resistant microtubules than control cells. Image by Jennifer Keynton.

Live time-lapse imaging of GFP–tubulin expressing epithelial cells depleted for EB2 show a marked reduction in microtubule dynamics (by J.Gadsby). Goldspink et al 2013. J Cell Sci. 126: 4000-4014

Control – EB2 present

Control – EB2 present

siRNA EB2 – no EB2

siRNA EB2 – no EB2

Non-centrosomal apico-basal microtubules originate from the centrosome

Confocal and electron microscope analyses of Nocodazole induced regrowth in MDCK and inner ear cells show that the apico-basal microtubule bundles in polarised epithelial cells originate from the centrosome.

Mt regrowth after Nocodazole removal showing centrosomal nucleation and Mt elongation towards the cortex prior to apico-basal array formation. Images by Gemma Bellett

Mt regrowth after Nocodazole removal showing centrosomal nucleation and Mt elongation towards the cortex prior to apico-basal array formation. Images by Gemma Bellett

Microtubules elongate from the centrosome towards the cell cortex following Nocodazole removal and an extended radial array initially forms. Bellett et al.2009. Cell Motil Cytoskel. 66:893-908

Formation of apico-basal arrays involves microtubule plus-end and minus-end capture at adherens junctions

Microtubule plus-ends target adherens junctions with EB1 and CLIP-170 co-localising with b-catenin and dynein clusters at the junction sites.

Extended radial microtubule (green) array forms following Nocodazole removal with EB1 (red) comets targeting junctions. Image by Gemma Bellett

Extended radial microtubule (green) array forms following Nocodazole removal with EB1 (red) comets targeting junctions. Image by Gemma Bellett

The extended radial array is likely to be an important intermediate step in the assembly of apico-basal microtubule arrays.

The minus-ends of the apico-basal microtubules associate with apical adherens junctions

TEM of polarised MDCK cell with microtubule minus-end (arrow) closely associated with apical adherens junction. Scale bar 100nm. Image by Gemma Bellett

TEM of polarised MDCK cell with microtubule minus-end (arrow) closely associated with apical adherens junction. Scale bar 100nm. Image by Gemma Bellett

Release and Capture Model for Apico-basal Microtubule Bundle Formation

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EB2 expression (+EB2) maintains dynamic microtubules during early stages of differentiation

  • Centrosome nucleated microtubules with EB2 along the lattice are captured at adherens junctions.
  • Cortical dynein associated with adherens junctions mediates microtubule release from the centrosome and facilitates microtubule translocation and downwards pulling.

Down-regulation of EB2 (-EB2) facilitates bundle formation

  • Formin nucleated actin filaments elongate towards the cell base
  • Actin filaments guide microtubules downwards facilitated by EB1 and ACF7.

See Bellett et al. 2009 Cell Motil Cytoskel 66:893-908 and Goldspink et al 2013. J Cell Sci. 126: 4000-4014

Centrosomal reorganisation during epithelial differentiation

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Live time-lapse imaging of GFP-ninein expressing epithelial cells show release of ninein from the centrosome.

See Moss et al. 2007. J Cell Sci. 120: 3064-3074

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Double transfection of GFP-ninein (pink) and YFP--tubulin (blue) showing bidirectional movement of ninein along microtubules.

Immuno-gold localises ninein along centrosome anchored microtubules.

Immuno-gold localises ninein along centrosome anchored microtubules.

Ninein relocates to apical non-centrosomal sites associated with adherens junctions during epithelial differentiation

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Polarised MDCK cells showing apico-basal microtubules (green) and ninein (yellow) at the centrosome and at apical non-centrosomal sites.

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Ninein (green) is relocated to apical non-centrosomal sites in inner pillar cells during inner ear development. γ-Tubulin (pink) remains concentrated at the centrosome.

See Moss et al. 2007. J Cell Sci. 120: 3064-3074

Computer Modeling of Microtubules

As microtubule dynamics change, so does their organisation.

compmod

We use computer models to investigate this process which produce images such as this. The models  are then compared to microtubules in real cells.