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Removal of MAP4 from microtubules in vivo produces no observable phenotype at the cellular level
X M Wang1, J G Peloquin, Y Zhai
1Laboratory of Molecular Biology, University of Wisconsin-Madison 53706, USA.
Abstract:
Microtubule-associated protein 4 (MAP4) promotes MT assembly in vitro and is localized along MTs in vivo. These results and the fact that MAP4 is the major MAP in nonneuronal cells suggest that MAP4's normal functions may include the stabilization of MTs in situ. To understand MAP4 function in vivo, we produced a blocking antibody (Ab) to prevent MAP4 binding to MTs. The COOH-terminal MT binding domain of MAP4 was expressed in Escherichia coli as a glutathione transferase fusion protein and was injected into rabbits to produce an antiserum that was then affinity purified and shown to be monospecific for MAP4. This Ab blocked > 95% of MAP4 binding to MTs in an in vitro assay. Microinjection of the affinity purified Ab into human fibroblasts and monkey epithelial cells abolished MAP4 binding to MTs as assayed with a rat polyclonal antibody against the NH2-terminal projection domain of MAP4. The removal of MAP4 from MTs was accompanied by its sequestration into visible MAP4-Ab immunocomplexes. However, the MT network appeared normal. Tubulin photoactivation and nocodazole sensitivity assays indicated that MT dynamics were not altered detectably by the removal of MAP4 from the MTs. Cells progressed to mitosis with morphologically normal spindles in the absence of MAP4 binding to MTs. Depleting MAP4 from MTs also did not affect the state of posttranslational modifications of tubulin subunits. Further, no perturbations of MT-dependent organelle distribution were detected. We conclude that the association of MAP4 with MTs is not essential for MT assembly or for the MT-based functions in cultured cells that we could assay. A significant role for MAP4 is not excluded by these results, however, as MAP4 may be a component of a functionally redundant system.
Insights
Microtubule-associated protein 4 (MAP4) binding to microtubules is not essential for cell functions like assembly or mitosis. MAP4 may play a role in a redundant system, as its removal did not impact microtubule dynamics or cell division.
Area of Science:
- Cell Biology
- Molecular Biology
- Cytoskeleton Dynamics
Background:
- Microtubule-associated protein 4 (MAP4) is a major microtubule-associated protein in nonneuronal cells.
- MAP4 promotes microtubule assembly in vitro and localizes to microtubules in vivo.
- Its function in stabilizing microtubules in situ is suggested but not fully understood.
Purpose of the Study:
- To investigate the in vivo function of MAP4 by disrupting its binding to microtubules.
- To determine if MAP4 is essential for microtubule assembly, dynamics, and cell division.
Main Methods:
- Production of a monospecific blocking antibody against the microtubule-binding domain of MAP4.
- Microinjection of the antibody into human fibroblasts and monkey epithelial cells.
- Assays for MAP4 binding, microtubule network integrity, tubulin dynamics, mitosis, tubulin posttranslational modifications, and organelle distribution.
Main Results:
- The antibody effectively blocked >95% of MAP4 binding to microtubules in vitro and in vivo.
- Removal of MAP4 from microtubules did not alter the microtubule network appearance or dynamics.
- Cells completed mitosis with normal spindles, and tubulin modifications and organelle distribution remained unaffected.
Conclusions:
- MAP4 association with microtubules is not essential for microtubule assembly or key microtubule-based functions in cultured cells.
- MAP4 might be part of a functionally redundant system, explaining its dispensability in these assays.