Fluorescent Probes For Real-time Quantitative Analysis Of Microtubule Dynamics

Microtubule as one of the major cytoskeletal components in eukaryotes plays an important role in many cellular activities.The microtubules are highly dynamic in cells,which are essential for cell division,cell migration,cell polarity and other activities.Real-time monitoring microtubule behaviors are helpful for better understanding of regulatory mechanisms governing microtubule architecture and microtubule-based activities.However,the precise molecular mechanism underlying the regulation of microtubule dynamics,especially how tubulin isotypes and post-translational modifications regulate microtubule dynamics,remain largely unclear.In vitro assays that combine total internal reflection fluorescence(TIRF)microscopy and reconstituted microtubules have provided molecular insights into the principles of microtubule dynamics and the roles of microtubule-associated proteins(MAPs).This technical method requires fluorescent labeling of tubulin in advance;however,most of the tubulin after fluorescent labeling loses polymerizing competence into microtubules,leading to an extremely low yield of fluorescently labeled-tubulin.It is thus demanded to prepare a large amount of tubulin for fluorescent labeling of tubulin.The current microtubule probes are designed and developed based on microtubuletargeting agent,which interferes with the dynamics of microtubules,limiting their application for microtubule dynamic determination.To this end,we chose peptides that bind to microtubules moderately as the recognition unit for the development of microtubule probes.In order to avoid interference with the dynamics of microtubules,we choose peptides with moderate affinity as the recognition unit of microtubule probes.The peptide fluorescent probe developed in this work can specifically label microtubules,which can replace fluorphoreconjugated tubulin for in vitro microtubule reconstitution.This allows the use of limited-source samples such as mice and cultured cells to investigate the regulatory mechanism of the tubulin code.Firstly,two commonly used phage libraries,Ph.D.-12 and Ph.D.-7 were selected.By panning through the assembled microtubules and purified tubulin for four rounds,some repetitive peptides were identified from these two libraries,and the peptides with the highest frequency were selected as candidate recognition units for the development of microtubule probes.Secondly,we selected FITC as the fluorescent group for the development of probe,and introduced aminoacetic acid to stabilize the coupling of the fluorescent group.After synthesizing such a primary probe,a pilot study was carried out.We found that the small peptide fragments with the highest frequency from the two phage libraries could not label microtubules.Regarding that microtubule tails contain a lot of negatively-charged glutamic acid aminos,we added 2,4,or 6 positively-charged arginines to the peptide and found that the peptide with 6 additional arginines was able to label microtubules.In addition,we also tested TAMRA,CY3,and CY5 fluorescent dyes and found that FITC-,CY3-and CY5-conjugated peptide but not TAMRA-conjugated peptide can ideally label microtubules.To sum up,we have successfully developed a multicolor fluorescent probe that can label microtubules in vitro under a total internal reflection fluorescence microscope.Next,the characteristics of peptide probes were analyzed.Through fluorescence photo-bleaching recovery experiments,we found that the peptide probe was resistant to photo-bleaching compared with commonly used fluorephore-conjugated tubulin.The peptide probe can label microtubules immediately after its addition,indicating the peptide probe is an add-and-use probe.In addition,we found that the peptide probe label can be washed out easily,making it possible to re-label microtubules with another fluorescent peptide probe.In addition,we also found that a relatively low concentration of peptide probe was able to label microtubules with a good signal to noise ratio.Then,we confirmed that peptide probes could also label dynamic microtubules.Compared with the standard used fluorephore-coujugated tubulin,the peptide probe did not interfere with the dynamics of microtubules.We purified several microtubule-associated proteins,including DCX,Kinesin-1,MCAK,ch TOG,and EB1 and revealed that the peptide probe did not interfere with the interactions of these microtubule-associated proteins with microtubules.In addition,we found that the peptide probe neither interfered with the sliding of Kinesin-1 along microtubules,nor interfered with microtubule regulatory function of MCAK,ch TOG,and EB1 proteins,indicating that the peptide probe can be used to study the regulation mechanism of microtubule dynamics.Finally,we used the peptide probe to analyze the regulation of microtubule dynamics by acetylation.We purified tubulin from HDAC6 depleted He La cells,and then analyzed the impact of HDAC6 regulated deacetylation modification on microtubule dynamics.Our results show that the peptide probe could be used for in vitro reconstitution experiments from limited-source samples,which solves the technical bottleneck that the generation of fluorophore conjugated tubulin requires a large amount of tubulin.In addition,attempts to develop fluorescent probes for imaging microtubules in living cells were made.By screening small peptide fragments that recognize microtubules in living cells,we found that 126 amino acid peptides derived from Tau was able to label microtubules in living cells.This peptide needs to be further optimized in the future.We will try to reduce the length of the peptide and improve its specificity.In summary,the in vitro peptide probe developed in this work can be used for in vitro microtubule reconstitution study.Compared with the currently commonly used fluorephoreconjugated tubulin,the probe has several advantages including photo-stability,ready-to-use,easy wash out and flexibility.The probe can be used to study the dynamics of microtubules and the regulation of microtubule-related proteins on the dynamics of microtubules.Importantly,this probe solves the technical bottleneck of the current fluorephore-conjugated tubulin generation that requires a large amount of tubulin in advance,and is suitable for studies with limited-source samples.Our work provides a powerful tool for in-depth understanding of the regulation mechanism of tubulin code on microtubule dynamics and microtubule-related cell activities.