Two lines of evi­dence indi­cate that micro­tubule tips approach focal adhe­sions at a range close enough for the pre­cise exhange of mol­e­c­u­lar sig­nals. The first evi­dence comes from the use of evanes­cent wave microscopy (Toomre and Manstein, 2001).

If a beam of light illu­mi­nates a trans­par­ent, reflect­ing sur­face at the angle of total inter­nal reflec­tion, an evanes­cent wave pen­e­trates above the sur­face to a depth of around 100200nm. With liv­ing cells grow­ing on a glass cov­er­slip, the illu­mi­na­tion is then restricted to the ven­tral sur­face, which includes the sub­strate adhe­sion sites.

Evanes­cent wave microscopy of cells labelled with GFP-tubulin shows that micro­tubules dip down into the evanes­cent wave as they grow towards the cell periph­ery:

Evanes­cent wave microscopy of a fibrob­last express­ing GFP-tubulin. By this tech­nique, the sam­ple is illu­mi­nated with excit­ing light in a layer only 100200nm above the sub­strate. The appear­ance of micro­tubules only at the cell periph­ery indi­cates that micro­tubules dip down towards the ven­tral cell sur­face in these periph­eral regions. Note that some micro­tubules fol­low iden­ti­cal tracks. (From Krylyshk­ina et al., 2003)

Dou­ble label­ing of cells for an adhe­sion com­po­nent (zyxin) and micro­tubules shows that the dip­ping down of micro­tubules cor­re­lates with their tar­get­ing of adhe­sion sites:

Evanes­cent wave microscopy of a fibrob­last express­ing GFP-tubulin and Ds-Red zyxin and imaged simul­ta­ne­ously in the green and red flu­o­res­cent chan­nels. Both pseudo colour and a black and white super­im­po­si­tion of images are shown. Where micro­tubules dip down towards the ven­tral cell sur­face, they tar­get adhe­sion sites. (From Krylyshk­ina et al., 2003)

The inten­sity of the evanes­cent wave decreases expo­nen­tially away from the sub­strate and the inten­sity of flu­o­res­cence of the micro­tubule tips gives a mea­sure of their depth of pen­e­tra­tion into the evanes­cent wave. Cal­cu­la­tions show that the micro­tubules approach the ven­tral cell sur­face within a range of 50nm. We return later to this phe­nom­e­non in the con­text of the com­plex of pro­teins found at micro­tubule tips.

A sec­ond indi­ca­tion of an inti­mate cross-talk between micro­tubules and focal adhe­sions is indi­cated by the abil­ity of adhe­sion sites to cap­ture depoly­meris­ing micro­tubules and to tem­porar­ily sta­bilise them. This is illus­trated in the fol­low­ing video: in this exper­i­ment, the cell was exposed dur­ing the sequence to noco­da­zole to depoly­merise micro­tubules. Fol­low­ing addi­tion of the drug (at the point of loss of focus), micro­tubules start to shorten. One of them (T) is sub­se­quently cap­tured dur­ing short­en­ing at an adhe­sion site (V) and stops depoly­meris­ing there for sev­eral min­utes (real time) before finally shrink­ing into the cell body (Kave­rina et al., 1998):

Adhe­sion sites can cap­ture micro­tubules. Video shows the periph­eral region of a fibrob­last that was co-injected with cy-3 tubu­lin and rhodamine-vinculin. Dur­ing the video, noco­da­zole was added to the medium to ini­ti­ate the depoly­meri­sa­tion of micro­tubules (addi­tion at point of defo­cus). A shrink­ing micro­tubule (T) is cap­tu­terd at a focal adhe­sion (V) and tem­porar­ily sta­bilised at the adhe­sion against depoly­meri­sa­tion. (From Kave­rina et al., 1998)

What is the con­se­quence of the tar­get­ing of adhe­sion sites by micro­tubules? Dif­fer­ent exper­i­ments sug­gest that micro­tubules retard the growth or stim­u­late the dis­as­sem­bly of focal adhe­sion sites.

A first exam­ple of the influ­ence of micro­tubules on adhe­sion dynam­ics is shown in the movie below. Here the for­ma­tion and turnover of adhe­sions were mon­i­tored in cells that were set­tling and spread­ing on a sub­strate. A con­trol cell (left) is com­pared with a cell that was pre­treated and main­tained in noco­da­zole to depoly­merise all micro­tubules. As the con­trol cell spreads, the first adhe­sions formed at the cell perime­ter (marked by arrow­heads) are dis­solved and replaced by new adhe­sions estab­lished at a larger radius. In the cell lack­ing micro­tubules (right) the early adhe­sion do not dis­solve, but just elon­gate as the cell radius increases. This lack of turnover of adhe­sions in the absence of micro­tubules retards spread­ing.

Micro­tubules are required to pro­mote adhe­sion site turnover dur­ing the spread­ing of cells on a sub­strate. Video pair shows two fish fibrob­lasts dur­ing the early stages of spread­ing on a sub­strate (both cells were trans­fected with GFP-zyxin to mark adhe­sion sites). A con­trol cell (with a nor­mal com­ple­ment of micro­tubules) is com­pared with a cell with­out micro­tubules (obtained by pre­treat­ment and incu­ba­tion dur­ing spread­ing in noco­da­zole). Note the dif­fer­ence in turnover of the adhe­sion sites marked by arrow­heads in the two sit­u­a­tions. Early adhe­sion sites in the con­trol cell turnover dur­ing spread­ing, to be replaced by new adhe­sions at a larger radius, whereas those in the nocodazole-treated cell per­sist and elon­gate.

A sec­ond exam­ple is shown below. In this exper­i­ment the desta­bil­is­ing effect of micro­tubules on adhe­sion is shown. The edge of a cell was exposed to noco­da­zole through a micronee­dle to depoly­merise micro­tubules locally. Adhe­sion sites marked by vin­culin pop­u­late the cell edge. The video shows the result of remov­ing the noco­da­zole to allow repoly­meri­sa­tion of the micro­tubules. Note that the micro­tubules grow into the adhe­sion sites at the cell periph­ery and that this cor­re­lates with the release of the adhe­sions from the sub­strate.

Micro­tubule tar­get­ing pro­motes the release of adhe­sions from the sub­strate. In this exper­i­ment, a fish fibrobast was injected with cy-3 tubu­lin and rho­damine vin­culin and then exposed to noco­da­zole through a micropipette to depoly­merise micro­tubules locally. The video shows the events after removal of noco­da­zole. Note that the micro­tubules grow to the cell periph­ery and tar­get the periph­eral adhe­sion sites (arrow­heads). Tar­get­ing is fol­lowed by the release of the adhe­sions and retrac­tion of the cell edge. (From Kave­rina et al., 1999)

A third exam­ple is shown for a motile cell in the next two fig­ures: the overview movie shows a fish fibrob­last pro­trud­ing on a broad front and retract­ing at the rear and flanks. Retrac­tion occurs in con­cert with the dis­so­ci­a­tion from the sub­strate of periph­eral focal adhe­sions. Details of the retract­ing flank are shown in the inset. Note that the dis­so­ci­a­tion of the adhe­sion sites (marked by arrow­heads) is pre­ceded and accom­pa­nied by the mul­ti­ple tar­get­ing of the adhe­sion sites by micro­tubules.

Microtubule-adhesion site tar­get­ing in a mov­ing cell. Video shows a fish fibrob­last that was injected with cy-3 tubu­lin and rho­damine vin­culin. Note pro­tru­sion of the cell front and the retrac­tion of the flanks and rear. Details in the inset are shown in the movie below.

Inset region from the pre­vi­ous movie, show­ing details of microtubule-adhesion site tar­get­ing. Selected adhe­sion sites are indi­cated by arrow­heads. Note that the retrac­tion of the adhe­sion sites is pre­ceded and accom­pa­nied by mul­ti­ple tar­get­ing events by micro­tubules. (From Kave­rina et al., 1999)

A fur­ther exam­ple of the dis­so­lu­tion of adhe­sion foci fol­low­ing mul­ti­ple tar­get­ing by micro­tubules is shown in the movie below:

Dis­so­lu­tion of adhe­sion sites fol­low­ing tar­get­ing by micro­tubules. Video shows a region of a cell that was trans­fected with GFP-tubulin and Ds-Red zyxin. The two adhe­sion sites cir­cled dis­ap­pear after mul­ti­ple tar­get­ing events by micro­tubules. (Video pro­duced by Irina Kave­rina)

Related Pub­li­ca­tions

  • Kave­rina, I., Rot­tner, K., Small, J. V. (1998). Tar­get­ing, cap­ture, and sta­bi­liza­tion of micro­tubules at early focal adhe­sions. J. Cell Biol. 142, 181190. PDF
  • Kave­rina, I., Krylyshk­ina, O., Small, J. V. (1999). Micro­tubule tar­get­ing of sub­strate con­tacts pro­motes their relax­ation and dis­so­ci­a­tion. J. Cell Biol. 146, 10331044. PDF
  • Krylyshk­ina, O., Ander­son, K. I., Kave­rina, I., Upmann, I., Manstein, D. J., Small, J. V., Toomre, D. K. (2003). Nanome­ter tar­get­ing of micro­tubules to focal adhe­sions. J. Cell Biol. 161, 853859. PDF 
  • Toomre, D. and Manstein, D. J. (2001). Light­ing up the cell sur­face with evanes­cent wave microscopy. Trends Cell. Biol. 11, 298303. NCBI PubMed

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