Subramanian, R., Ti, S.C., Tan, L., Darst, S.A., Kapoor, T.M. (2013), Marking and measuring single microtubules by PRC1 and kinesin-4. Cell, 145, 377-90. (Highlighted in Developmental Cell, 26, 118)
Nanometer-sized proteins generate micron-sized tags at distinct cellular sites to spatially regulate function across biology. Typically, such tags depend on proteins specifically recognizing structural features (e.g. DNA sequence at telomeres) that remain stable for periods that are much longer than protein association and activity timescales. However, it is unclear how proteins can selectively tag microtubules, which turnover on timescales faster than that of typical biochemical mechanisms (e.g. phosphorylation). To address this long-standing question, we focused on PRC1 and kinesin-4, two microtubule-associated proteins required to assemble the spindle midzone, a specialized array of microtubule bundles that keeps segregated chromosomes apart and positions the division plane in human cells. Using TIRF microscopy-based assays we show that purified PRC1 and kinesin-4 form a complex that can generate micron-sized tags at microtubule plus-ends, the more dynamic end of the bio-polymer. Remarkably, the size of these tags is proportional to microtubule length. We determined PRC1’s crystal structure to map protein-protein interactions and designed constructs to dissect the biochemical mechanism for microtubule length-dependent tagging. Our findings indicate that PRC1/kinesin-4 complexes bind stochastically along microtubules and can ‘walk’ several microns towards the plus-end. Many of these complexes reach the filament end and accumulate to form a tag. Steady-state tag size is achieved when accumulation and dissociation rates match. As the available binding sites for PRC1/kinesin-4 scale with microtubule length, tag length is proportional to that of the filament. Importantly, similar to what we observe with purified proteins, PRC1 generates microtubule length-dependent tags in dividing cells. Our findings reveal how molecular recognition and active transport can be combined to allow ensembles of nanometer-sized proteins to effectively measure micron-scale features within dynamic filament networks. We propose that PRC1/kinesin-4 allows differential tagging and regulation of dynamic microtubules based on their length to organize macromolecular assemblies, such as spindle midzones.
Nanometer-sized proteins generate micron-sized tags at distinct cellular sites to spatially regulate function across biology. Typically, such tags depend on proteins specifically recognizing structural features (e.g. DNA sequence at telomeres) that remain stable for periods that are much longer than protein association and activity timescales. However, it is unclear how proteins can selectively tag microtubules, which turnover on timescales faster than that of typical biochemical mechanisms (e.g. phosphorylation). To address this long-standing question, we focused on PRC1 and kinesin-4, two microtubule-associated proteins required to assemble the spindle midzone, a specialized array of microtubule bundles that keeps segregated chromosomes apart and positions the division plane in human cells. Using TIRF microscopy-based assays we show that purified PRC1 and kinesin-4 form a complex that can generate micron-sized tags at microtubule plus-ends, the more dynamic end of the bio-polymer. Remarkably, the size of these tags is proportional to microtubule length. We determined PRC1’s crystal structure to map protein-protein interactions and designed constructs to dissect the biochemical mechanism for microtubule length-dependent tagging. Our findings indicate that PRC1/kinesin-4 complexes bind stochastically along microtubules and can ‘walk’ several microns towards the plus-end. Many of these complexes reach the filament end and accumulate to form a tag. Steady-state tag size is achieved when accumulation and dissociation rates match. As the available binding sites for PRC1/kinesin-4 scale with microtubule length, tag length is proportional to that of the filament. Importantly, similar to what we observe with purified proteins, PRC1 generates microtubule length-dependent tags in dividing cells. Our findings reveal how molecular recognition and active transport can be combined to allow ensembles of nanometer-sized proteins to effectively measure micron-scale features within dynamic filament networks. We propose that PRC1/kinesin-4 allows differential tagging and regulation of dynamic microtubules based on their length to organize macromolecular assemblies, such as spindle midzones.
