Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 1999 Apr;10(4):1105-18.
doi: 10.1091/mbc.10.4.1105.

Detyrosination of tubulin regulates the interaction of intermediate filaments with microtubules in vivo via a kinesin-dependent mechanism

G Kreitzer  1 , G Liao, G G Gundersen
Affiliations
Free PMC article

Detyrosination of tubulin regulates the interaction of intermediate filaments with microtubules in vivo via a kinesin-dependent mechanism

G Kreitzer et al. Mol Biol Cell. 1999 Apr.
Free PMC article

Abstract

Posttranslationally modified forms of tubulin accumulate in the subset of stabilized microtubules (MTs) in cells but are not themselves involved in generating MT stability. We showed previously that stabilized, detyrosinated (Glu) MTs function to localize vimentin intermediate filaments (IFs) in fibroblasts. To determine whether tubulin detyrosination or MT stability is the critical element in the preferential association of IFs with Glu MTs, we microinjected nonpolymerizable Glu tubulin into cells. If detyrosination is critical, then soluble Glu tubulin should be a competitive inhibitor of the IF-MT interaction. Before microinjection, Glu tubulin was rendered nonpolymerizable and nontyrosinatable by treatment with iodoacetamide (IAA). Microinjected IAA-Glu tubulin disrupted the interaction of IFs with MTs, as assayed by the collapse of IFs to a perinuclear location, and had no detectable effect on the array of Glu or tyrosinated MTs in cells. Conversely, neither IAA-tyrosinated tubulin nor untreated Glu tubulin, which assembled into MTs, caused collapse of IFs when microinjected. The epitope on Glu tubulin responsible for interfering with the Glu MT-IF interaction was mapped by microinjecting tubulin fragments of alpha-tubulin. The 14-kDa C-terminal fragment of Glu tubulin (alpha-C Glu) induced IF collapse, whereas the 36-kDa N-terminal fragment of alpha-tubulin did not alter the IF array. The epitope required more than the detyrosination site at the C terminus, because a short peptide (a 7-mer) mimicking the C terminus of Glu tubulin did not disrupt the IF distribution. We previously showed that kinesin may mediate the interaction of Glu MTs and IFs. In this study we found that kinesin binding to MTs in vitro was inhibited by the same reagents (i.e., IAA-Glu tubulin and alpha-C Glu) that disrupted the IF-Glu MT interaction in vivo. These results demonstrate for the first time that tubulin detyrosination functions as a signal for the recruitment of IFs to MTs via a mechanism that is likely to involve kinesin.

PubMed Disclaimer

Figures

Figure 1
Figure 1
Preparation of pure Glu and Tyr tubulin. Tubulin, purified from brain tissue (left) and from HeLa cells (right), was treated with CPA to remove the C-terminal tyrosine residues. Samples of the CPA-treated (+) or untreated (−) preparations were analyzed by Western blot to determine the levels of Glu and Tyr tubulin. Blots were loaded with 0.5 μg of tubulin per lane and were reacted with antibodies specific to Glu tubulin (Glu) and to Tyr tubulin (Tyr). Only the region corresponding to α-tubulin is shown. Note that brain tubulin is a mixture of Glu and Tyr tubulin before CPA treatment (∼50:50) whereas HeLa tubulin is predominantly Tyr tubulin (92% Tyr vs. 8% Glu). CPA treatment of brain or HeLa tubulin almost completely eliminated reactivity with the Tyr tubulin antibody and increased reactivity with the Glu tubulin antibody.
Figure 2
Figure 2
Microinjected IAA-Glu tubulin does not polymerize and is not retyrosinated. Wound-edge 3T3 cells were injected with 140 μM IAA-treated Glu tubulin (a and b) or 100 μM untreated Glu tubulin (c and d) and incubated for 5 or 120 min at 37°C before fixation. Human IgG (2 mg/ml) was coinjected as a marker. Cells were immunostained for Glu tubulin (a–d), human IgG (insets in b and d), and Tyr tubulin. Injected cells are marked with asterisks at the cell periphery. Note that after 5 min both IAA-Glu tubulin and untreated Glu tubulin in injected cells are detected as diffuse immunofluorescence in the cytoplasm with anti-Glu tubulin antibodies. The filamentous Glu MT staining observed is attributable to the staining of endogenous Glu MTs. After 120 min only those cells injected with IAA-Glu tubulin are identifiable by a diffuse, anti-Glu tubulin immunofluorescent staining pattern (b), indicative of the presence of nonpolymerizable Glu tubulin that is not retyrosinated after microinjection into cells. Conversely, cells injected with untreated Glu tubulin must be identified by human IgG immunoreactivity (d) because most of the injected Glu tubulin has been retyrosinated (our unpublished results) and incorporated into the MT network. Bar, 10 μm.
Figure 3
Figure 3
Microinjection of nonpolymerizable, IAA-Glu tubulin causes collapse of the IF network. 3T3 cells at the edge of an in vitro wound were injected with IAA-treated Glu tubulin (from calf brain) at 140 μM (a–c) or at 70 μM (d–f) or with untreated Glu tubulin at 100 μM (g–i). A marker protein, human IgG (2 mg/ml), was coinjected in all cases. Cells were fixed 2 h after injection and immunofluorescently stained to reveal vimentin (a, d, and g), Glu tubulin (b, e, and h), human IgG (c and i), and Tyr tubulin (f). The asterisks in a, d, and g indicate the cell periphery of the injected cell (the human IgG marker is not shown for d–f). Note that in the cells injected with IAA-Glu the IFs are collapsed around the nucleus and do not extend to the cell edge, whereas in cells injected with untreated, assembly-competent Glu tubulin the IFs remain extended in the cytoplasm. Microinjection of human IgG alone had no apparent effect on the distributions of either the MTs or the IFs (our unpublished results). Bar, 10 μm.
Figure 4
Figure 4
Microinjected IAA-HeLa Glu tubulin, but not IAA-HeLa Tyr tubulin, induces collapse of the IF network. 3T3 cells at the edge of a wound were injected with 100 μM IAA-HeLa Glu tubulin (a and b) or 140 μM IAA-HeLa Tyr tubulin (c and d), fixed 2 h later, and immunofluorescently stained as described above. Human IgG (2 mg/ml) was coinjected as a marker to identify the injected cells. Shown are the vimentin IFs (a and c) and the injected human IgG marker (b and d). The asterisks indicate the peripheral edge of injected cells in a and c. Bar, 10 μm.
Figure 5
Figure 5
Effect of microinjected monomeric tubulins on IF distribution. Wound-edge 3T3 cells were microinjected with polymerization-competent brain Glu tubulin, IAA-treated brain Glu tubulin, IAA-treated HeLa Tyr tubulin, or IAA-treated HeLa Glu tubulin. The IF collapse in each injected cell was determined visually by fluorescence microscopy. IFs were considered to be collapsed when the entire IF network extended <30% of the distance from the nucleus to the leading edge of the cell. Brain Glu and IAA-Glu tubulins were microinjected at 100 μM; HeLa IAA-Glu and IAA-Tyr tubulins were microinjected at 70 μM. Data are pooled from two or more experiments that gave similar results. For uninjected cells (UNINJ.), n = 200; for brain Glu tubulin, n = 51; for brain IAA-Glu tubulin, n = 410; for HeLa IAA-Tyr tubulin, n = 121; and for HeLa IAA-Glu tubulin, n = 265.
Figure 6
Figure 6
C-terminal fragments of α-tubulin induce collapse of IFs. Wound-edge cells were microinjected with N-terminal (α-N) or C-terminal (α-C and α-C Glu) α-tubulin trypsin fragments, incubated for 2 h at 37°C, fixed, and immunofluorescently stained for vimentin IFs (a, c, and e), human IgG marker (b and d), Glu tubulin (f), and Tyr tubulin. IFs are unaltered in cells injected with 70 μM α-N (a and b) but are collapsed to a perinuclear region in cells injected with 145 μM α-C (c and d) or 235 μM α-C Glu (e and f). Asterisks denote the peripheral edge of injected cells. Bar, 10 μm.
Figure 7
Figure 7
Effect of nonpolymerizable IAA-tubulins and α-tubulin fragments and peptides on binding of kinesin to MTs. The kinesin head (K394) was incubated with MTs in the absence or presence of IAA-HeLa Glu or IAA-HeLa Tyr tubulin (A), α-tubulin fragments (B; α-C, α-N, and α-C Glu), or C-terminal peptides of α-tubulin (B), was pelleted by centrifugation, and was analyzed by SDS-PAGE and Coomassie staining. Pellet and supernatant (Spt) fractions are shown in A, whereas only pellet fractions are shown in B. Bands marked T and K are tubulin and the kinesin head K394, respectively. Note, in A, that little kinesin head is detected in the MT pellet in the sample incubated with IAA-HeLa Glu tubulin (kinesin head is instead recovered in the supernatant), but significant kinesin head is recovered in MT pellets in the sample incubated with IAA-HeLa Tyr tubulin. The additional tubulin recovered in the supernatants in the IAA-HeLa Glu and Tyr tubulin samples reflects the added nonpolymerizable tubulin. Con, control.
Figure 8
Figure 8
Effectiveness of nonpolymerizable IAA-tubulin and tubulin fragments and peptides in collapsing IFs in vivo and in inhibiting kinesin binding to MTs in vitro. For determination of the effect on IF distribution in vivo, wound-edge 3T3 cells were microinjected with IAA-HeLa Glu tubulin (Glu), IAA-HeLa Tyr tubulin (Tyr), α-tubulin fragments (α-C, α-N, and α-C Glu), the C-terminal 7-mer peptide of Glu tubulin (7-mer), and the C-terminal 8-mer peptide of Tyr tubulin (8-mer). Injected cells were assayed for IF collapse as described in Figure 5. IAA-HeLa Glu tubulin and IAA-HeLa Tyr tubulin were microinjected at 70 μM; α-N, α-C, and α-C Glu were microinjected at 170, 145, and 235 μM, respectively; and C-terminal Glu and Tyr tubulin peptides (7-mer and 8-mer) were microinjected at 20 mM. All concentrations refer to concentrations in the microinjection needle; the final concentration in the cells would be approximately one-tenth of that microinjected (see text). For determination of the effect on kinesin binding to MTs, the conventional kinesin head (K394) (1.4 μM) was incubated with taxol-stabilized brain MTs (2 μM) in the presence of the same tubulin and tubulin fragments described above, and samples were incubated as described in MATERIALS AND METHODS. IAA-HeLa Glu or IAA-HeLa Tyr tubulin and α-tubulin fragments were used at 14 μM, and the C-terminal peptides of Glu and Tyr tubulin were used at 150 μM. The amount of K394 that cosedimented with MTs and the level of tubulin in the MT pellets were quantified by SDS-PAGE and densitometry analysis. The amount of K394 cosedimenting with MTs was normalized to the level of tubulin in the MT pellet and compared with that of the control sample to obtain the inhibition of kinesin binding (%).
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Alexander JE, Hunt DF, Lee MK, Shabanowitz J, Michel H, Berlin SC, MacDonald TL, Sundberg RJ, Rebhun LI, Frankfurter A. Characterization of posttranslational modifications in neuron-specific class III β-tubulin by mass spectrometry. Proc Natl Acad Sci USA. 1991;88:4685–4689. - PMC - PubMed
    1. Baas PW, Black MM. Individual microtubules in the axon consist of domains that differ in both composition and stability. J Cell Biol. 1990;111:495–509. - PMC - PubMed
    1. Ball EH, Singer SJ. Mitochondria are associated with microtubules and not with intermediate filaments in cultured fibroblasts. Proc Natl Acad Sci USA. 1982;79:123–126. - PMC - PubMed
    1. Barra HS, Rodriguez JA, Arce CA, Caputto R. A soluble preparation from rat brain that incorporates into its own proteins [14C]-arginine by a ribonuclease-sensitive system and [14C]-tyrosine by a ribonuclease-insensitive system. J Neurochem. 1973;20:97–108. - PubMed
    1. Breitling F, Little M. Carboxy-terminal regions on the surface of tubulin and microtubules. Epitope locations of YOL1/34, DM1A and DM1B. J Mol Biol. 1986;189:367–370. - PubMed

Publication types

MeSH terms

LinkOut - more resources

-