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Detection of GTP-tubulin by immunofluorescence in mammalian cells. Cultured cells were processed for hMB11 immunostaining ( 9 ) and microtubules were stained with the hF2C antibody (MDA- MB231 cells) or by GFP-tubulin expres- sion (HeLa and Ptk2 cells). Boxed regions are shown enlarged (×5) below. Some microtubule ends were stained by hMB11 (white arrowheads); others were not (open arrowheads). hMB11 also detected GTP- tubulin dots inside the polymers (open arrows). In HeLa or Ptk2, extended stretches corresponding to microtubule bundling were also strongly stained (white arrows). Scale bar, 10 m m.
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Microtubules display dynamic instability, with alternating phases of growth and shrinkage separated by catastrophe and rescue
events. The guanosine triphosphate (GTP) cap at the growing end of microtubules, whose presence is essential to prevent microtubule
catastrophes in vitro, has been difficult to observe in vivo. We selected a recombinant anti...
Contexts in source publication
Context 1
... are highly dynamic tubulin M polymers lular organization that are and essential cell division. for intracel- They display a dynamic instability, with alternating phases of growth and shrinkage separated by catastrophe and rescue events ( 1 , 2 ). Tubulin polymerizes in a guanosine triphosphate (GTP) – bound form and hydrolyzes GTP in the polymer with a slight delay. This creates a GTP cap at the growing end of microtubules ( 2 – 4 ). Loss of the GTP cap promotes catastrophic events, whereas microtubule rescues result from uncharacterized stochastic events. Even though the characteristics of the GTP cap have been well studied in vitro, the evidence that such a cap exists in vivo is lacking, essen- tially because no antibodies specific for the GTP- bound conformation of tubulin are available. The GTP-bound tubulin dimer is in a straighter conformation than the guanosine diphosphate (GDP) – bound dimer ( 5 ), and even when constrained in the lattice, GDP-tubulin does not have the same conformation as GTP-tubulin ( 6 , 7 ). This suggests that it should be possible to make conformational antibodies that specifically recognize GTP-bound tubulin in the polymer. Conformational antibodies specific for GTP-bound Rab6 were selected in vitro by antibody phage display ( 8 ). Here, we selected a recombinant antibody specific for the GTP-bound conformation of tubulin in the polymer. We used this antibody to localize GTP-tubulin in cellular microtubules. We screened a phage display library of recom- binant scFv (single-chain fragment variable) against guanosine 5 ́- O -(3 ́-thiotriphosphate) (GTP- g -S) – loaded tubulin and selected a se- ries of recombinant antibodies to tubulin ( ) (fig. S1). One scFv, named hMB11 (scFv MB11 fused to the Fc domain of human immuno- globulin G), was found to be conformation- specific. It did not recognize denatured tubulin by immunoblotting and seemed not to bind to native nonpolymerized tubulin. However, hMB11 cosedimented specifically with microtubules polymerized in the presence of guanylyl 5 ′ -( b , g -methylenediphosphonate) (GMPCPP), a nonhydrolyzable GTP analog, and not with control microtubules assembled in the presence of GTP (Fig. 1A). In this experiment, low concentrations of taxol (0.1 to 1 m M) were used to prevent depolymerization of control microtubules. When a higher concentration of taxol was used, hMB11 bound to both control and GMPCPP microtubules (Fig. 1, B and C),which suggests that it recognized a conformation and not the nucleotide itself. We then used hMB11 to stain by immunofluorescence a mixture of microtubules polymerized from pure tubulin in the presence of GTP or GMPCPP (Fig. 1D). Under these conditions, hMB11 stained only GMPCPP microtubules [representing 68.6 T 17.3% (SD) of MB11-positive microtubules] and not control microtubules (1.8 T 0.9%). The remaining 29.7 T 16.6% were bundles of both GMPCPP and control microtubules. Despite varying experi- mental conditions, not all GMPCPP-containing microtubules were stained by MB11, which suggests that some microtubules possessed conformational defects under these conditions. in cellular microtubules. We next used hMB11 to localize GTP-tubulin in cellular microtubules by immunofluorescence. Because of its conformational binding, hMB11 staining was very sensitive to structural alterations occurring after fixation ( 10 ). It was best to use unfixed cells permeabilized in the presence of glycer- ol and/or low taxol concentration to prevent microtubule depolymerization. In three repre- sentative cell lines (HeLa, Ptk2, and MDA- MB231), hMB11 stained the tips of only a fraction of microtubules (Fig. 2, white arrowheads representing 63 T 4.5% of visible ends), whereas other microtubule ends were unstained (Fig. 2, open arrowheads). This was expected because the GTP-cap model proposes that only microtubules growing at the time of staining should be capped with GTP-tubulin. The observed proportion was very close to the 60% of growing microtubules identified in interphase cells ( 11 ). In addition to the microtubule tip staining predicted by the GTP-cap model, we also observed an unexpected GTP-tubulin staining. First, hMB11 labeled long internal stretches in areas where microtubules formed bundles (Fig. 2, white arrows), although not all bundles were positive. The occurrences of these stretches depended on the cell line used. It is not known whether the GTP domains of microtubules are prone to bundling (as observed upon long incubation with taxol; see fig. S2) or whether microtubules retain a GTP conformation due to bundling and/or to specific binding proteins. Second, hMB11 detected dots along individual microtubules, which we have termed “ GTP remnants, ” that looked randomly distributed (Fig. 2, open arrows). GTP caps and GTP remnants were also detected in mitotic cells and were more abun- dant in spindle than in astral microtubules (fig. S3). Microtubules polymerized in vitro from GTP-tubulin were similarly stained by hMB11 at some of their ends and on discrete internal regions (Fig. 3A). To determine whether labeled ends could correspond to GTP caps, we stained microtubule asters that had polymerized from centrosomes for a short period of time. As predicted by the GTP-cap model, the majority of microtubule plus ends (73% of 226 microtubules in 22 asters) were labeled (Fig. 3B, arrows). Intriguingly, and as shown above, a few discrete internal microtubule regions were also decorated. One possibility is that hMB11 may be directed against a domain in tubulin that would face the lumen of the tube and thus only be accessible at plus ends and on random structural defects along microtubules. This seems unlikely, however, because hMB11 decorated microtubules all along their length when expressed intracellularly while fused to mCherry (fig. S4). We propose that hMB11 stains GTP-bound or GDP – inorganic phosphate (GDP-Pi) – bound tubulin dimers that have been trapped in small regions of the microtubules. A molecular me- chanical model indeed predicted that the presence of GTP dimers in the lattice would only locally perturb the microtubule structure ( 12 ). Experimentally, GTP or GDP-Pi tubulin have been detected in microtubules ( 13 – 16 ). However, more recent studies have failed to detect GTP-bound or GDP-Pi – bound subunits in microtubules, and the presence of very small caps has been proposed ( , , , ), although this has recently been challenged ( 19 ). In any case, only a small fraction of GTP-tubulin is present inside the polymer. crotubule rescue domains. The presence of GTP-tubulin conformation in microtubules suggests a model for dynamic instability (Fig. 4A) that would provide some mechanistic basis to the seemingly stochastic rescue events. In this model, GTP hydrolysis is not always com- plete and some tubulin dimers persist in a GTP conformation in the polymer. Upon depolymerization, these GTP remnants will be- come exposed. If GTP hydrolysis does not resume, any remnant as small as a single tubulin layer ( 4 ) may behave as a polymerization- prone GTP cap, thereby promoting microtubule rescue. The GTP remnants may explain the frequent rescue events observed when polymerizing microtubules experience short- ening ( 19 ). Note that growing GTP caps are structurally shaped as open sheets, whereas un- covered internal GTP remnants may exhibit blunt ends. To test our model, we analyzed the dynamic behavior of microtubules in Ptk2 cells stably expressing a GFP (green fluorescent protein) – tubulin fusion protein and performed retrospective staining of GTP remnants. Figure 4B and movie S1 show such a sequence in which various events can be identified in par- ticular microtubule rescues (arrows). The polymerizing microtubule exhibited a GTP cap (Fig. 4B, white arrowhead), as did more than 80% of the microtubules that were growing at the time of cell extraction (Fig. 4C). A large fraction of the rescue events recorded [88.8 T 7.8% (SEM); 38 rescues, 35 microtubules, eight cells] occurred at locations where GTP remnants were retrospectively identified, thus supporting the GTP remnant model (see kymographs, Fig. 4B). A Monte Carlo simulation predicted that only 7.77 T 1.53% coincidence would be expected to occur by chance ( 9 ) (Fig. 4C and table S1). GTP remnant dis- tribution was roughly proportional to rescue frequency (see the comparison between RPE1 and Ptk2 cell lines, Fig. 4C), even though only one-third of GTP remnants seemed to rescue microtubules efficiently. In addition, GTP remnants could be found in newly polymerized portions of microtubules that had never encountered a rescue event (fig. S5), which suggests that the GTP remnants are most prob- ably the cause rather than the consequence of rescue. On the basis of these findings, we wrote simulation software to visualize the different models of microtubule dynamic instability ( 9 ) (MTsimul v1.4; fig. S6 and movie S2). Ac- cording to the GTP-cap model, rescue depends on the probability of GDP tubulin present at the tip of the depolymerizing microtubule to start polymerizing again. Under the GTP-remnant model, rescues are linked to the probability of GTP hydrolysis. This implies that rescue locations are memorized in the polymer during the seconds or minutes before actual rescues, allowing cells to predetermine their microtubule life span. Factors may exist that would regulate GTP-remnant frequency and thus microtubule ...
Context 2
... are highly dynamic tubulin M polymers lular organization that are and essential cell division. for intracel- They display a dynamic instability, with alternating phases of growth and shrinkage separated by catastrophe and rescue events ( 1 , 2 ). Tubulin polymerizes in a guanosine triphosphate (GTP) – bound form and hydrolyzes GTP in the polymer with a slight delay. This creates a GTP cap at the growing end of microtubules ( 2 – 4 ). Loss of the GTP cap promotes catastrophic events, whereas microtubule rescues result from uncharacterized stochastic events. Even though the characteristics of the GTP cap have been well studied in vitro, the evidence that such a cap exists in vivo is lacking, essen- tially because no antibodies specific for the GTP- bound conformation of tubulin are available. The GTP-bound tubulin dimer is in a straighter conformation than the guanosine diphosphate (GDP) – bound dimer ( 5 ), and even when constrained in the lattice, GDP-tubulin does not have the same conformation as GTP-tubulin ( 6 , 7 ). This suggests that it should be possible to make conformational antibodies that specifically recognize GTP-bound tubulin in the polymer. Conformational antibodies specific for GTP-bound Rab6 were selected in vitro by antibody phage display ( 8 ). Here, we selected a recombinant antibody specific for the GTP-bound conformation of tubulin in the polymer. We used this antibody to localize GTP-tubulin in cellular microtubules. We screened a phage display library of recom- binant scFv (single-chain fragment variable) against guanosine 5 ́- O -(3 ́-thiotriphosphate) (GTP- g -S) – loaded tubulin and selected a se- ries of recombinant antibodies to tubulin ( ) (fig. S1). One scFv, named hMB11 (scFv MB11 fused to the Fc domain of human immuno- globulin G), was found to be conformation- specific. It did not recognize denatured tubulin by immunoblotting and seemed not to bind to native nonpolymerized tubulin. However, hMB11 cosedimented specifically with microtubules polymerized in the presence of guanylyl 5 ′ -( b , g -methylenediphosphonate) (GMPCPP), a nonhydrolyzable GTP analog, and not with control microtubules assembled in the presence of GTP (Fig. 1A). In this experiment, low concentrations of taxol (0.1 to 1 m M) were used to prevent depolymerization of control microtubules. When a higher concentration of taxol was used, hMB11 bound to both control and GMPCPP microtubules (Fig. 1, B and C),which suggests that it recognized a conformation and not the nucleotide itself. We then used hMB11 to stain by immunofluorescence a mixture of microtubules polymerized from pure tubulin in the presence of GTP or GMPCPP (Fig. 1D). Under these conditions, hMB11 stained only GMPCPP microtubules [representing 68.6 T 17.3% (SD) of MB11-positive microtubules] and not control microtubules (1.8 T 0.9%). The remaining 29.7 T 16.6% were bundles of both GMPCPP and control microtubules. Despite varying experi- mental conditions, not all GMPCPP-containing microtubules were stained by MB11, which suggests that some microtubules possessed conformational defects under these conditions. in cellular microtubules. We next used hMB11 to localize GTP-tubulin in cellular microtubules by immunofluorescence. Because of its conformational binding, hMB11 staining was very sensitive to structural alterations occurring after fixation ( 10 ). It was best to use unfixed cells permeabilized in the presence of glycer- ol and/or low taxol concentration to prevent microtubule depolymerization. In three repre- sentative cell lines (HeLa, Ptk2, and MDA- MB231), hMB11 stained the tips of only a fraction of microtubules (Fig. 2, white arrowheads representing 63 T 4.5% of visible ends), whereas other microtubule ends were unstained (Fig. 2, open arrowheads). This was expected because the GTP-cap model proposes that only microtubules growing at the time of staining should be capped with GTP-tubulin. The observed proportion was very close to the 60% of growing microtubules identified in interphase cells ( 11 ). In addition to the microtubule tip staining predicted by the GTP-cap model, we also observed an unexpected GTP-tubulin staining. First, hMB11 labeled long internal stretches in areas where microtubules formed bundles (Fig. 2, white arrows), although not all bundles were positive. The occurrences of these stretches depended on the cell line used. It is not known whether the GTP domains of microtubules are prone to bundling (as observed upon long incubation with taxol; see fig. S2) or whether microtubules retain a GTP conformation due to bundling and/or to specific binding proteins. Second, hMB11 detected dots along individual microtubules, which we have termed “ GTP remnants, ” that looked randomly distributed (Fig. 2, open arrows). GTP caps and GTP remnants were also detected in mitotic cells and were more abun- dant in spindle than in astral microtubules (fig. S3). Microtubules polymerized in vitro from GTP-tubulin were similarly stained by hMB11 at some of their ends and on discrete internal regions (Fig. 3A). To determine whether labeled ends could correspond to GTP caps, we stained microtubule asters that had polymerized from centrosomes for a short period of time. As predicted by the GTP-cap model, the majority of microtubule plus ends (73% of 226 microtubules in 22 asters) were labeled (Fig. 3B, arrows). Intriguingly, and as shown above, a few discrete internal microtubule regions were also decorated. One possibility is that hMB11 may be directed against a domain in tubulin that would face the lumen of the tube and thus only be accessible at plus ends and on random structural defects along microtubules. This seems unlikely, however, because hMB11 decorated microtubules all along their length when expressed intracellularly while fused to mCherry (fig. S4). We propose that hMB11 stains GTP-bound or GDP – inorganic phosphate (GDP-Pi) – bound tubulin dimers that have been trapped in small regions of the microtubules. A molecular me- chanical model indeed predicted that the presence of GTP dimers in the lattice would only locally perturb the microtubule structure ( 12 ). Experimentally, GTP or GDP-Pi tubulin have been detected in microtubules ( 13 – 16 ). However, more recent studies have failed to detect GTP-bound or GDP-Pi – bound subunits in microtubules, and the presence of very small caps has been proposed ( , , , ), although this has recently been challenged ( 19 ). In any case, only a small fraction of GTP-tubulin is present inside the polymer. crotubule rescue domains. The presence of GTP-tubulin conformation in microtubules suggests a model for dynamic instability (Fig. 4A) that would provide some mechanistic basis to the seemingly stochastic rescue events. In this model, GTP hydrolysis is not always com- plete and some tubulin dimers persist in a GTP conformation in the polymer. Upon depolymerization, these GTP remnants will be- come exposed. If GTP hydrolysis does not resume, any remnant as small as a single tubulin layer ( 4 ) may behave as a polymerization- prone GTP cap, thereby promoting microtubule rescue. The GTP remnants may explain the frequent rescue events observed when polymerizing microtubules experience short- ening ( 19 ). Note that growing GTP caps are structurally shaped as open sheets, whereas un- covered internal GTP remnants may exhibit blunt ends. To test our model, we analyzed the dynamic behavior of microtubules in Ptk2 cells stably expressing a GFP (green fluorescent protein) – tubulin fusion protein and performed retrospective staining of GTP remnants. Figure 4B and movie S1 show such a sequence in which various events can be identified in par- ticular microtubule rescues (arrows). The polymerizing microtubule exhibited a GTP cap (Fig. 4B, white arrowhead), as did more than 80% of the microtubules that were growing at the time of cell extraction (Fig. 4C). A large fraction of the rescue events recorded [88.8 T 7.8% (SEM); 38 rescues, 35 microtubules, eight cells] occurred at locations where GTP remnants were retrospectively identified, thus supporting the GTP remnant model (see kymographs, Fig. 4B). A Monte Carlo simulation predicted that only 7.77 T 1.53% coincidence would be expected to occur by chance ( 9 ) (Fig. 4C and table S1). GTP remnant dis- tribution was roughly proportional to rescue frequency (see the comparison between RPE1 and Ptk2 cell lines, Fig. 4C), even though only one-third of GTP remnants seemed to rescue microtubules efficiently. In addition, GTP remnants could be found in newly polymerized portions of microtubules that had never encountered a rescue event (fig. S5), which suggests that the GTP remnants are most prob- ably the cause rather than the consequence of rescue. On the basis of these findings, we wrote simulation software to visualize the different models of microtubule dynamic instability ( 9 ) (MTsimul v1.4; fig. S6 and movie S2). Ac- cording to the GTP-cap model, rescue depends on the probability of GDP tubulin present at the tip of the depolymerizing microtubule to start polymerizing again. Under the GTP-remnant model, rescues are linked to the probability of GTP hydrolysis. This implies that rescue locations are memorized in the polymer during the seconds or minutes before actual rescues, allowing cells to predetermine their microtubule life span. Factors may exist that would regulate GTP-remnant frequency and thus microtubule ...
Context 3
... are highly dynamic tubulin M polymers lular organization that are and essential cell division. for intracel- They display a dynamic instability, with alternating phases of growth and shrinkage separated by catastrophe and rescue events ( 1 , 2 ). Tubulin polymerizes in a guanosine triphosphate (GTP) – bound form and hydrolyzes GTP in the polymer with a slight delay. This creates a GTP cap at the growing end of microtubules ( 2 – 4 ). Loss of the GTP cap promotes catastrophic events, whereas microtubule rescues result from uncharacterized stochastic events. Even though the characteristics of the GTP cap have been well studied in vitro, the evidence that such a cap exists in vivo is lacking, essen- tially because no antibodies specific for the GTP- bound conformation of tubulin are available. The GTP-bound tubulin dimer is in a straighter conformation than the guanosine diphosphate (GDP) – bound dimer ( 5 ), and even when constrained in the lattice, GDP-tubulin does not have the same conformation as GTP-tubulin ( 6 , 7 ). This suggests that it should be possible to make conformational antibodies that specifically recognize GTP-bound tubulin in the polymer. Conformational antibodies specific for GTP-bound Rab6 were selected in vitro by antibody phage display ( 8 ). Here, we selected a recombinant antibody specific for the GTP-bound conformation of tubulin in the polymer. We used this antibody to localize GTP-tubulin in cellular microtubules. We screened a phage display library of recom- binant scFv (single-chain fragment variable) against guanosine 5 ́- O -(3 ́-thiotriphosphate) (GTP- g -S) – loaded tubulin and selected a se- ries of recombinant antibodies to tubulin ( ) (fig. S1). One scFv, named hMB11 (scFv MB11 fused to the Fc domain of human immuno- globulin G), was found to be conformation- specific. It did not recognize denatured tubulin by immunoblotting and seemed not to bind to native nonpolymerized tubulin. However, hMB11 cosedimented specifically with microtubules polymerized in the presence of guanylyl 5 ′ -( b , g -methylenediphosphonate) (GMPCPP), a nonhydrolyzable GTP analog, and not with control microtubules assembled in the presence of GTP (Fig. 1A). In this experiment, low concentrations of taxol (0.1 to 1 m M) were used to prevent depolymerization of control microtubules. When a higher concentration of taxol was used, hMB11 bound to both control and GMPCPP microtubules (Fig. 1, B and C),which suggests that it recognized a conformation and not the nucleotide itself. We then used hMB11 to stain by immunofluorescence a mixture of microtubules polymerized from pure tubulin in the presence of GTP or GMPCPP (Fig. 1D). Under these conditions, hMB11 stained only GMPCPP microtubules [representing 68.6 T 17.3% (SD) of MB11-positive microtubules] and not control microtubules (1.8 T 0.9%). The remaining 29.7 T 16.6% were bundles of both GMPCPP and control microtubules. Despite varying experi- mental conditions, not all GMPCPP-containing microtubules were stained by MB11, which suggests that some microtubules possessed conformational defects under these conditions. in cellular microtubules. We next used hMB11 to localize GTP-tubulin in cellular microtubules by immunofluorescence. Because of its conformational binding, hMB11 staining was very sensitive to structural alterations occurring after fixation ( 10 ). It was best to use unfixed cells permeabilized in the presence of glycer- ol and/or low taxol concentration to prevent microtubule depolymerization. In three repre- sentative cell lines (HeLa, Ptk2, and MDA- MB231), hMB11 stained the tips of only a fraction of microtubules (Fig. 2, white arrowheads representing 63 T 4.5% of visible ends), whereas other microtubule ends were unstained (Fig. 2, open arrowheads). This was expected because the GTP-cap model proposes that only microtubules growing at the time of staining should be capped with GTP-tubulin. The observed proportion was very close to the 60% of growing microtubules identified in interphase cells ( 11 ). In addition to the microtubule tip staining predicted by the GTP-cap model, we also observed an unexpected GTP-tubulin staining. First, hMB11 labeled long internal stretches in areas where microtubules formed bundles (Fig. 2, white arrows), although not all bundles were positive. The occurrences of these stretches depended on the cell line used. It is not known whether the GTP domains of microtubules are prone to bundling (as observed upon long incubation with taxol; see fig. S2) or whether microtubules retain a GTP conformation due to bundling and/or to specific binding proteins. Second, hMB11 detected dots along individual microtubules, which we have termed “ GTP remnants, ” that looked randomly distributed (Fig. 2, open arrows). GTP caps and GTP remnants were also detected in mitotic cells and were more abun- dant in spindle than in astral microtubules (fig. S3). Microtubules polymerized in vitro from GTP-tubulin were similarly stained by hMB11 at some of their ends and on discrete internal regions (Fig. 3A). To determine whether labeled ends could correspond to GTP caps, we stained microtubule asters that had polymerized from centrosomes for a short period of time. As predicted by the GTP-cap model, the majority of microtubule plus ends (73% of 226 microtubules in 22 asters) were labeled (Fig. 3B, arrows). Intriguingly, and as shown above, a few discrete internal microtubule regions were also decorated. One possibility is that hMB11 may be directed against a domain in tubulin that would face the lumen of the tube and thus only be accessible at plus ends and on random structural defects along microtubules. This seems unlikely, however, because hMB11 decorated microtubules all along their length when expressed intracellularly while fused to mCherry (fig. S4). We propose that hMB11 stains GTP-bound or GDP – inorganic phosphate (GDP-Pi) – bound tubulin dimers that have been trapped in small regions of the microtubules. A molecular me- chanical model indeed predicted that the presence of GTP dimers in the lattice would only locally perturb the microtubule structure ( 12 ). Experimentally, GTP or GDP-Pi tubulin have been detected in microtubules ( 13 – 16 ). However, more recent studies have failed to detect GTP-bound or GDP-Pi – bound subunits in microtubules, and the presence of very small caps has been proposed ( , , , ), although this has recently been challenged ( 19 ). In any case, only a small fraction of GTP-tubulin is present inside the polymer. crotubule rescue domains. The presence of GTP-tubulin conformation in microtubules suggests a model for dynamic instability (Fig. 4A) that would provide some mechanistic basis to the seemingly stochastic rescue events. In this model, GTP hydrolysis is not always com- plete and some tubulin dimers persist in a GTP conformation in the polymer. Upon depolymerization, these GTP remnants will be- come exposed. If GTP hydrolysis does not resume, any remnant as small as a single tubulin layer ( 4 ) may behave as a polymerization- prone GTP cap, thereby promoting microtubule rescue. The GTP remnants may explain the frequent rescue events observed when polymerizing microtubules experience short- ening ( 19 ). Note that growing GTP caps are structurally shaped as open sheets, whereas un- covered internal GTP remnants may exhibit blunt ends. To test our model, we analyzed the dynamic behavior of microtubules in Ptk2 cells stably expressing a GFP (green fluorescent protein) – tubulin fusion protein and performed retrospective staining of GTP remnants. Figure 4B and movie S1 show such a sequence in which various events can be identified in par- ticular microtubule rescues (arrows). The polymerizing microtubule exhibited a GTP cap (Fig. 4B, white arrowhead), as did more than 80% of the microtubules that were growing at the time of cell extraction (Fig. 4C). A large fraction of the rescue events recorded [88.8 T 7.8% (SEM); 38 rescues, 35 microtubules, eight cells] occurred at locations where GTP remnants were retrospectively identified, thus supporting the GTP remnant model (see kymographs, Fig. 4B). A Monte Carlo simulation predicted that only 7.77 T 1.53% coincidence would be expected to occur by chance ( 9 ) (Fig. 4C and table S1). GTP remnant dis- tribution was roughly proportional to rescue frequency (see the comparison between RPE1 and Ptk2 cell lines, Fig. 4C), even though only one-third of GTP remnants seemed to rescue microtubules efficiently. In addition, GTP remnants could be found in newly polymerized portions of microtubules that had never encountered a rescue event (fig. S5), which suggests that the GTP remnants are most prob- ably the cause rather than the consequence of rescue. On the basis of these findings, we wrote simulation software to visualize the different models of microtubule dynamic instability ( 9 ) (MTsimul v1.4; fig. S6 and movie S2). Ac- cording to the GTP-cap model, rescue depends on the probability of GDP tubulin present at the tip of the depolymerizing microtubule to start polymerizing again. Under the GTP-remnant model, rescues are linked to the probability of GTP hydrolysis. This implies that rescue locations are memorized in the polymer during the seconds or minutes before actual rescues, allowing cells to predetermine their microtubule life span. Factors may exist that would regulate GTP-remnant frequency and thus microtubule ...
Context 4
... are highly dynamic tubulin M polymers lular organization that are and essential cell division. for intracel- They display a dynamic instability, with alternating phases of growth and shrinkage separated by catastrophe and rescue events ( 1 , 2 ). Tubulin polymerizes in a guanosine triphosphate (GTP) – bound form and hydrolyzes GTP in the polymer with a slight delay. This creates a GTP cap at the growing end of microtubules ( 2 – 4 ). Loss of the GTP cap promotes catastrophic events, whereas microtubule rescues result from uncharacterized stochastic events. Even though the characteristics of the GTP cap have been well studied in vitro, the evidence that such a cap exists in vivo is lacking, essen- tially because no antibodies specific for the GTP- bound conformation of tubulin are available. The GTP-bound tubulin dimer is in a straighter conformation than the guanosine diphosphate (GDP) – bound dimer ( 5 ), and even when constrained in the lattice, GDP-tubulin does not have the same conformation as GTP-tubulin ( 6 , 7 ). This suggests that it should be possible to make conformational antibodies that specifically recognize GTP-bound tubulin in the polymer. Conformational antibodies specific for GTP-bound Rab6 were selected in vitro by antibody phage display ( 8 ). Here, we selected a recombinant antibody specific for the GTP-bound conformation of tubulin in the polymer. We used this antibody to localize GTP-tubulin in cellular microtubules. We screened a phage display library of recom- binant scFv (single-chain fragment variable) against guanosine 5 ́- O -(3 ́-thiotriphosphate) (GTP- g -S) – loaded tubulin and selected a se- ries of recombinant antibodies to tubulin ( ) (fig. S1). One scFv, named hMB11 (scFv MB11 fused to the Fc domain of human immuno- globulin G), was found to be conformation- specific. It did not recognize denatured tubulin by immunoblotting and seemed not to bind to native nonpolymerized tubulin. However, hMB11 cosedimented specifically with microtubules polymerized in the presence of guanylyl 5 ′ -( b , g -methylenediphosphonate) (GMPCPP), a nonhydrolyzable GTP analog, and not with control microtubules assembled in the presence of GTP (Fig. 1A). In this experiment, low concentrations of taxol (0.1 to 1 m M) were used to prevent depolymerization of control microtubules. When a higher concentration of taxol was used, hMB11 bound to both control and GMPCPP microtubules (Fig. 1, B and C),which suggests that it recognized a conformation and not the nucleotide itself. We then used hMB11 to stain by immunofluorescence a mixture of microtubules polymerized from pure tubulin in the presence of GTP or GMPCPP (Fig. 1D). Under these conditions, hMB11 stained only GMPCPP microtubules [representing 68.6 T 17.3% (SD) of MB11-positive microtubules] and not control microtubules (1.8 T 0.9%). The remaining 29.7 T 16.6% were bundles of both GMPCPP and control microtubules. Despite varying experi- mental conditions, not all GMPCPP-containing microtubules were stained by MB11, which suggests that some microtubules possessed conformational defects under these conditions. in cellular microtubules. We next used hMB11 to localize GTP-tubulin in cellular microtubules by immunofluorescence. Because of its conformational binding, hMB11 staining was very sensitive to structural alterations occurring after fixation ( 10 ). It was best to use unfixed cells permeabilized in the presence of glycer- ol and/or low taxol concentration to prevent microtubule depolymerization. In three repre- sentative cell lines (HeLa, Ptk2, and MDA- MB231), hMB11 stained the tips of only a fraction of microtubules (Fig. 2, white arrowheads representing 63 T 4.5% of visible ends), whereas other microtubule ends were unstained (Fig. 2, open arrowheads). This was expected because the GTP-cap model proposes that only microtubules growing at the time of staining should be capped with GTP-tubulin. The observed proportion was very close to the 60% of growing microtubules identified in interphase cells ( 11 ). In addition to the microtubule tip staining predicted by the GTP-cap model, we also observed an unexpected GTP-tubulin staining. First, hMB11 labeled long internal stretches in areas where microtubules formed bundles (Fig. 2, white arrows), although not all bundles were positive. The occurrences of these stretches depended on the cell line used. It is not known whether the GTP domains of microtubules are prone to bundling (as observed upon long incubation with taxol; see fig. S2) or whether microtubules retain a GTP conformation due to bundling and/or to specific binding proteins. Second, hMB11 detected dots along individual microtubules, which we have termed “ GTP remnants, ” that looked randomly distributed (Fig. 2, open arrows). GTP caps and GTP remnants were also detected in mitotic cells and were more abun- dant in spindle than in astral microtubules (fig. S3). Microtubules polymerized in vitro from GTP-tubulin were similarly stained by hMB11 at some of their ends and on discrete internal regions (Fig. 3A). To determine whether labeled ends could correspond to GTP caps, we stained microtubule asters that had polymerized from centrosomes for a short period of time. As predicted by the GTP-cap model, the majority of microtubule plus ends (73% of 226 microtubules in 22 asters) were labeled (Fig. 3B, arrows). Intriguingly, and as shown above, a few discrete internal microtubule regions were also decorated. One possibility is that hMB11 may be directed against a domain in tubulin that would face the lumen of the tube and thus only be accessible at plus ends and on random structural defects along microtubules. This seems unlikely, however, because hMB11 decorated microtubules all along their length when expressed intracellularly while fused to mCherry (fig. S4). We propose that hMB11 stains GTP-bound or GDP – inorganic phosphate (GDP-Pi) – bound tubulin dimers that have been trapped in small regions of the microtubules. A molecular me- chanical model indeed predicted that the presence of GTP dimers in the lattice would only locally perturb the microtubule structure ( 12 ). Experimentally, GTP or GDP-Pi tubulin have been detected in microtubules ( 13 – 16 ). However, more recent studies have failed to detect GTP-bound or GDP-Pi – bound subunits in microtubules, and the presence of very small caps has been proposed ( , , , ), although this has recently been challenged ( 19 ). In any case, only a small fraction of GTP-tubulin is present inside the polymer. crotubule rescue domains. The presence of GTP-tubulin conformation in microtubules suggests a model for dynamic instability (Fig. 4A) that would provide some mechanistic basis to the seemingly stochastic rescue events. In this model, GTP hydrolysis is not always com- plete and some tubulin dimers persist in a GTP conformation in the polymer. Upon depolymerization, these GTP remnants will be- come exposed. If GTP hydrolysis does not resume, any remnant as small as a single tubulin layer ( 4 ) may behave as a polymerization- prone GTP cap, thereby promoting microtubule rescue. The GTP remnants may explain the frequent rescue events observed when polymerizing microtubules experience short- ening ( 19 ). Note that growing GTP caps are structurally shaped as open sheets, whereas un- covered internal GTP remnants may exhibit blunt ends. To test our model, we analyzed the dynamic behavior of microtubules in Ptk2 cells stably expressing a GFP (green fluorescent protein) – tubulin fusion protein and performed retrospective staining of GTP remnants. Figure 4B and movie S1 show such a sequence in which various events can be identified in par- ticular microtubule rescues (arrows). The polymerizing microtubule exhibited a GTP cap (Fig. 4B, white arrowhead), as did more than 80% of the microtubules that were growing at the time of cell extraction (Fig. 4C). A large fraction of the rescue events recorded [88.8 T 7.8% (SEM); 38 rescues, 35 microtubules, eight cells] occurred at locations where GTP remnants were retrospectively identified, thus supporting the GTP remnant model (see kymographs, Fig. 4B). A Monte Carlo simulation predicted that only 7.77 T 1.53% coincidence would be expected to occur by chance ( 9 ) (Fig. 4C and table S1). GTP remnant dis- tribution was roughly proportional to rescue frequency (see the comparison between RPE1 and Ptk2 cell lines, Fig. 4C), even though only one-third of GTP remnants seemed to rescue microtubules efficiently. In addition, GTP remnants could be found in newly polymerized portions of microtubules that had never encountered a rescue event (fig. S5), which suggests that the GTP remnants are most prob- ably the cause rather than the consequence of rescue. On the basis of these findings, we wrote simulation software to visualize the different models of microtubule dynamic instability ( 9 ) (MTsimul v1.4; fig. S6 and movie S2). Ac- cording to the GTP-cap model, rescue depends on the probability of GDP tubulin present at the tip of the depolymerizing microtubule to start polymerizing again. Under the GTP-remnant model, rescues are linked to the probability of GTP hydrolysis. This implies that rescue locations are memorized in the polymer during the seconds or minutes before actual rescues, allowing cells to predetermine their microtubule life span. Factors may exist that would regulate GTP-remnant frequency and thus microtubule ...
Citations
... The released GDP-tubulin dimers exchange their GDP with a GTP, regaining their ability to assemble. The prevailing model for rescue, i.e., the transition from shrinkage to growth, is that GTP-tubulin islands in the lattice buffer against depolymerization and promote growth (Dimitrov et al., 2008;Aumeier et al., 2016;Vemu et al., 2018). ...
... This work may thus resolve a question raised 15 years ago in the context of chemical kinetic and structural plasticity approaches to microtubule dynamics (Kueh and Mitchison, 2009). Dimitrov and colleagues (Dimitrov et al., 2008) had identified short stable segments in the middle of microtubules in vivo, and Kueh and Mitchison noted that it remained to be determined whether those stable segments contained GTP-tubulin (chemical kinetic model) or GDP-tubulin in an alternative state (structural plasticity model). Our data support the latter view. ...
... Second, there is the phenomenon of pausing, which has been observed in cells but not in vitro in the absence of external factors (Lieuvin et al., 1994;van Riel et al., 2017): our experiments show that GDP-tubulin incorporation at the plus end is sufficient to cause pauses. Third, GDP-tubulin islands could favor rescues or repairing damaged sites, as has been proposed for GTP-tubulin islands (Dimitrov et al., 2008;Schaedel et al., 2015;de Forges et al., 2016;Vemu et al., 2018;Andreu-Carbó et al., 2022). To our knowledge, none of these studies tested whether GDP-tubulin might also repair damaged lattice sites. ...
Microtubules are dynamic polymers that interconvert between phases of growth and shrinkage, yet they provide structural stability to cells. Growth involves hydrolysis of GTP-tubulin to GDP-tubulin, which releases energy that is stored within the microtubule lattice and destabilizes it; a GTP cap at microtubule ends is thought to prevent GDP subunits from rapidly dissociating and causing catastrophe. Here, using in vitro reconstitution assays, we show that GDP-tubulin, usually considered inactive, can itself assemble into microtubules, preferentially at the minus end, and promote persistent growth. GDP-tubulin-assembled microtubules are highly stable, displaying no detectable spontaneous shrinkage. Strikingly, islands of GDP-tubulin within dynamic microtubules stop shrinkage events and promote rescues. Microtubules thus possess an intrinsic capacity for stability, independent of accessory proteins. This finding provides novel mechanisms to explain microtubule dynamics.
... As a result, most tubulins in the middle region of a microtubule are thought to be in the unstable GDP-bound state, while freshly incorporated tubulin molecules at the microtubule tips hold GTP to form stable GTP caps. The detection of GTP-tubulins using conformationspecific antibodies further confirmed that the tip regions are in a distinct state (Dimitrov et al., 2008). Regulation of the GTP cap structure, the GTPase activity, and the synergy of MAPs and the microtubule lattice was thought to play a central role in controlling cell shapes and cellular components during cell division and locomotion (Zanic et al., 2013). ...
Microtubule cytoskeletons play pivotal roles in various cellular processes, including cell division and locomotion, by dynamically changing their length and distribution in cells through tubulin polymerization/depolymerization. Recent structural studies have revealed the polymorphic lattice structure of microtubules closely correlate with the microtubule dynamics, but the studies were limited to averaged structures. To reveal the transient and localized structures, such as GTP-cap, we developed several non-averaging methods for cryogenic electron tomography to precisely measure the longitudinal spacing and helical twisting of individual microtubule lattices at the tubulin subunit level. Our analysis revealed that polymerizing and depolymerizing ends share a similar structure with regards to lattice spacing. The most distinctive property specific to the polymerizing plus end was left-handed twisting in the inter-dimer interface, suggesting that the twisting might accelerate fast polymerization at the plus ends. Our analysis uncovered the heterogeneity of native microtubules and will be indispensable for the study of microtubules dynamics under physiological contexts or during specific cellular events.
... But does kinesin-1 also impact the distribution of these damages? To study the distribution of damage sites, we used a damage/repair site-specific antibody that detects tubulin conformational changes within the microtubule 50,51 . In HeLa cells the levels of damage/repair sites increased from the cell center towards the periphery (Fig. 1a, c). ...
... To address whether damage sites directly correlate with microtubule deacetylation, we analyzed the local acetylation levels around the damage/repair sites using the damage/repair site-specific antibody 50,51 . Figure 3e, f show that microtubule damage sites are embedded within stretches of deacetylated microtubules, with an average length of 0.76 ± 0.38 µm, consistent with the reported length of in vitro damage sites 40,44,46 . ...
The properties of single microtubules within the microtubule network can be modulated through post-translational modifications (PTMs), including acetylation within the lumen of microtubules. To access the lumen, the enzymes could enter through the microtubule ends and at damage sites along the microtubule shaft. Here we show that the acetylation profile depends on damage sites, which can be caused by the motor protein kinesin-1. Indeed, the entry of the deacetylase HDAC6 into the microtubule lumen can be modulated by kinesin-1-induced damage sites. In contrast, activity of the microtubule acetylase αTAT1 is independent of kinesin-1-caused shaft damage. On a cellular level, our results show that microtubule acetylation distributes in an exponential gradient. This gradient results from tight regulation of microtubule (de)acetylation and scales with the size of the cells. The control of shaft damage represents a mechanism to regulate PTMs inside the microtubule by giving access to the lumen.
... However, an early experiment with end-stabilized MTs by Dye et al. [8] clearly showed that the shaft may lose and incorporate tubulin dimers directly. Later it was shown that GTP dimers (or dimers in the GTP conformation) exist outside of the cap region [9], without a clear picture of how the GTP state could survive sufficiently long to be detectable in the shaft. A very recent series of experiments revealed that the shaft lattice exhibits a spontaneous dynamics, part of which is linked to lattice dislocations [10,11]. ...
Microtubules are key structural elements of living cells that are crucial for cell division, intracellular transport, and motility. Recent experiments have shown that microtubule-severing proteins and molecular motors stimulate the direct and localized incorporation of free tubulin into the shaft. However, a mechanistic picture of how microtubule-associated proteins affect the lattice is completely missing. Here we theoretically explore a potential mechanism of lattice turnover stimulated by processive molecular motors in which a weak transient destabilization of the lattice by the motor stepping promotes the formation of mobile vacancies. In the absence of free tubulin the defect rapidly propagates, leading to a complete fracture. In the presence of free tubulin, the motor walk induces a vacancy drift in the direction opposite of the motor walk. The drift is accompanied by the direct and localized incorporation of free tubulin along the trajectory of the vacancy. Our results are consistent with experiments and strongly suggest that a weak lattice-motor interaction is responsible for an augmented microtubule shaft plasticity.
... Microtubules polymerize at the plus-end via the incorporation of fresh guanosine triphosphate (GTP) to β-tubulin, which is hydrolyzed to guanosine diphosphate (GDP) in already incorporated tubulin dimers. However, GTP-bound tubulin dimers have also been described in the stable microtubule lattice [15][16][17] and are more enriched in axons than dendrites [17,18]. These so-named GTP islands protect microtubule depolymerization and promote self-repair [19,20] but also regulate the local conformation of tubulin to modulate the transport of mitochondria [18]. ...
The highly specialized structure and function of neurons depend on a sophisticated organization of the cytoskeleton, which supports a similarly sophisticated system to traffic organelles and cargo vesicles. Mitochondria sustain crucial functions by providing energy and buffering calcium where it is needed. Accordingly, the distribution of mitochondria is not even in neurons and is regulated by a dynamic balance between active transport and stable docking events. This system is finely tuned to respond to changes in environmental conditions and neuronal activity. In this review, we summarize the mechanisms by which mitochondria are selectively transported in different compartments, taking into account the structure of the cytoskeleton, the molecular motors and the metabolism of neurons. Remarkably, the motor proteins driving the mitochondrial transport in axons have been shown to also mediate their transfer between cells. This so-named intercellular transport of mitochondria is opening new exciting perspectives in the treatment of multiple diseases.
... Microtubules polymerize at the plus-end via incorporation of fresh GTP to β-tubulin, which is hydrolysed to GDP in already incorporated tubulin dimers. However, GTP-bound tubulin dimers have been described also in the stable microtubule lattice (15)(16)(17) and are more enriched in axons than dendrites (17,18). These so-named GTP islands protect microtubule depolymerisation and promote self-repair (19,20) but also to regulate the local conformation of tubulin to modulate the transport of mitochondria (18). ...
The highly specialized structure and function of neurons depend on a sophisticated organization of the cytoskeleton, which supports a similarly sophisticated system to traffic organelles and cargo vesicles. Mitochondria sustain crucial functions by providing energy and buffering calcium where is needed. Accordingly, the distribution of mitochondria is not even in neurons and is regulated by a dynamic balance between active transport and stable docking events. This system is finely tuned to respond to changes in environmental conditions and neuronal activity. In this review, we summarize the mechanisms by which mitochondria are selectively transported in different compartments taking into account the structure of the cytoskeleton, the molecular motors and the metabolism of neurons. Remarkably, the motor proteins driving the mitochondrial transport in axons have been shown to mediate also their transfer between cells. This so-named intercellular transport of mitochondria is opening new exciting perspectives in the treatment of multiple diseases.
... For example, segments of GTP-tubulin (i.e. GTP islands) can be observed within the lattice of GDPmicrotubules ( Fig. 1, thick outline) using the hMB11 antibody (Dimitrov et al., 2008). In addition, tubulin subunits within the lattice can be marked by post-translational modifications (PTMs) (Fig. 1, magenta circles) that are posited to encrypt spatial, temporal and functional information important for specific microtubule functions. ...
... The insertion of expanded GTP-tubulin subunits at sites of repair can also influence microtubule dynamics. These GTP-tubulin islands can serve as rescue sites during microtubule depolymerization (Dimitrov et al., 2008;Tropini et al., 2012;Aumeier et al., 2016;de Forges et al., 2016;Vemu et al., 2018;Schaedel et al., 2019;Bollinger et al., 2020;Rai et al., 2021), and thus a second output of kinesin-1-induced lattice damage could be an increase in microtubule length and overall density. Indeed, using dynamic microtubules, addition of kinesin-1 resulted in a concentration-dependent increase in rescue frequency and microtubule length (Andreu-Carbo et al., 2022;Budaitis et al., 2022). ...
Microtubules are critical for a variety of important functions in eukaryotic cells. During intracellular trafficking, molecular motor proteins of the kinesin superfamily drive the transport of cellular cargoes by stepping processively along the microtubule surface. Traditionally, the microtubule has been viewed as simply a track for kinesin motility. New work is challenging this classic view by showing that kinesin-1 and kinesin-4 proteins can induce conformational changes in tubulin subunits while they are stepping. These conformational changes appear to propagate along the microtubule such that the kinesins can work allosterically through the lattice to influence other proteins on the same track. Thus, the microtubule is a plastic medium through which motors and other microtubule-associated proteins (MAPs) can communicate. Furthermore, stepping kinesin-1 can damage the microtubule lattice. Damage can be repaired by the incorporation of new tubulin subunits, but too much damage leads to microtubule breakage and disassembly. Thus, the addition and loss of tubulin subunits are not restricted to the ends of the microtubule filament but rather, the lattice itself undergoes continuous repair and remodeling. This work leads to a new understanding of how kinesin motors and their microtubule tracks engage in allosteric interactions that are critical for normal cell physiology.
... The switch from growth to shrinkage is termed catastrophe and is thought to be due to the loss of a cap of GTP-tubulin at the growing end (2). The switch from shrinkage to growth is termed rescue and is thought to be due to reincorporation of GTP-tubulin during the shrinking process (3,4). These transitions, termed dynamic instability, control turnover of microtubule polymers (5,6) and allow microtubule tips to explore cytoplasmic space, to capture chromosomes during mitosis (7), to create pushing forces that position the mitotic spindle (8) and the nucleus (9), and to fill axons and dendrites during neuronal morphogenesis (10)(11)(12). ...
Microtubules are dynamic polymers that undergo stochastic transitions between growing and shrinking phases. The structural and chemical properties of these phases remain poorly understood. The transition from growth to shrinkage, termed catastrophe, is not a first-order reaction but is rather a multi-step process whose frequency increases with the growth time: the microtubule ages as the older microtubule tip becomes more unstable. Aging shows that the growing phase is not a single state but comprises several substates of increasing instability. To investigate whether the shrinking phase is also multi-state, we characterized the kinetics of microtubule shrinkage following catastrophe using an in vitro reconstitution assay with purified tubulins. We found that the shrinkage speed is highly variable across microtubules and that the shrinkage speed of individual microtubules slows down over time by as much as several fold. The shrinkage slowdown was observed in both fluorescently labeled and unlabeled microtubules as well as in microtubules polymerized from tubulin purified from different species, suggesting that the shrinkage slowdown is a general property of microtubules. These results indicate that microtubule shrinkage, like catastrophe, is time-dependent and that the shrinking microtubule tip passes through a succession of states of increasing stability. We hypothesize that the shrinkage slowdown is due to destabilizing events that took place during growth which led to multi-step catastrophe. This suggests that the aging associated with growth is also manifested during shrinkage with the older, more unstable growing tip being associated with a faster depolymerizing shrinking tip.
... To study whether microtubule deacetylation occurs at damage sites, we analyzed the local acetylation levels around the damage sites by using a damage/repair site-specific antibody 47,48 and co-stained for acetylation. In WT cells the levels of damage/repair sites increased from the cell center to the periphery (Fig. 6a). ...
The properties of single microtubules within the microtubule network can be modulated through posttranslational modifications (PTMs), including acetylation within the lumen of microtubules. To access the lumen, the enzymes could either enter through the microtubule ends or at damage sites along the microtubule shaft. Here we show that the acetylation profile depends on damage sites, which can be caused by the motor protein kinesin-1. Indeed, the entry of the deacetylase HDAC6 into the microtubule lumen depends on kinesin-1-induced damage sites. In contrast, activity of the microtubule acetylase αTAT1 is independent of kinesin-1 and shaft damage. On a cellular level, our results show that microtubule acetylation distributes in an exponential gradient. This gradient results from tight regulation of microtubule (de-)acetylation and scales with the size of the cells. The control of shaft damage represents a novel mechanism to regulate PTM inside the microtubule by giving access to the lumen.
... Molecular mechanism of microtubule polymerization appears complicated, including permanent and apparently random changes of protofilaments' ends at intervals between relatively slow growth and rapid shrinkage of microtubules (Kerssemakers et al. 2006;Schek et al. 2007;Gardner et al. 2008). It is assumed that stability of microtubules depend mainly on the stability of the cap, i.e., the GTP-tubulin form at the ends of these structures, and dynamics of the GTP-tubulin vs. GDP-tubulin conversion (Carlier et al. 1987;O'Brien et al. 1987;Bayley et al. 1990;Drechsel and Kirschner 1994;Caplow and Shanks 1996;Schek et al. 2007;Dimitrov et al. 2008). ...
Neurodegenerative diseases represent a large group of disorders characterized by gradual loss of neurons and functions of the central nervous systems. Their course is usually severe, leading to high morbidity and subsequent inability of patients to independent functioning. Vast majority of neurodegenerative diseases is currently untreatable, and only some symptomatic drugs are available which efficacy is usually very limited. To develop novel therapies for this group of diseases, it is crucial to understand their pathogenesis and to recognize factors which can influence the disease course. One of cellular structures which dysfunction appears to be relatively poorly understood in the light of neurodegenerative diseases is tubulin cytoskeleton. On the other hand, its changes, both structural and functional, can considerably influence cell physiology, leading to pathological processes occurring also in neurons. In this review, we summarize and discuss dysfunctions of tubulin cytoskeleton in various neurodegenerative diseases different than primary tubulinopathies (caused by mutations in genes encoding the components of the tubulin cytoskeleton), especially Alzheimer’s disease, Parkinson’s disease, Huntington’s disease, amyotrophic lateral sclerosis, prion diseases, and neuronopathic mucopolysaccharidoses. It is also proposed that correction of these disorders might attenuate the progress of specific diseases, thus, finding newly recognized molecular targets for potential drugs might become possible.
