Close

A live-cell marker to visualize the dynamics of stable microtubules

Klara I. Jansen, Mithila Burute, Lukas C. Kapitein

Preprint posted on June 23, 2021 https://www.biorxiv.org/content/10.1101/2021.06.23.449589v1

Rigor-2xmNeongreen: bringing stable microtubule dynamics to the fore.

Selected by Niveda Udaykumar

Background

The skeleton of a cell is its microtubule assembly, this underlies vital processes like cell division and intracellular transport. Various labeling studies have revealed that different microtubule types exist within a cell. Of these, the most widely studied are the dynamic microtubules. In this preprint, the authors instead aimed to facilitate the study of another microtubule subset: the stable microtubules.

These two types of microtubules differ in stability, distribution, and function: hence their names. The dynamic microtubules are prone to MT-destabilizing agents and are found uniformly distributed within the cell. In contrast, stable microtubules can withstand the effects of MT- destabilizing agents. This type is usually found in the leading edge of fibroblasts[1], and perinuclear areas in common cell lines like U2OS, HeLa, etc. The dynamic microtubule subset is well studied due to the wide availability of live-cell markers. However, stable microtubules have only been studied on fixed cells using immunocytochemistry, and the lack of a live-cell marker has limited the study of this subset of microtubules greatly.

To aid the direct visualization and study of stable microtubules, Jansen et.al. developed a live-cell marker, rigor-2xmNeongreen. This marker binds selectively to stable microtubules, facilitating studies on the dynamics and mechanisms of stable microtubules.

Key findings

Validation of rigor-2xmNeongreen as a potential live-cell marker for stable MTs:

The motor protein kinesin-1(hkif5b) moves specifically on stable microtubules[2,3,4]. A rigor mutant of this motor protein was developed by replacing glycine with alanine at the 234th position (G234A). This abolishes its ATPase activity and prevents its unbinding from the MTs[5]. This mutant can bind to acetylated stable MTs [4,6,7], a frequent post-translational modification observed in MTs.

A live-cell marker for stable microtubules was developed by fusing the G234A rigor mutant in tandem with a fluorophore, mNeongreen[8] (rigor-2xmNeongreen). Detection of rigor-2xmNeongreen expression in U2OS cells revealed that the marker could selectively bind to acetylated MTs, a proxy for stable MTs.

Fig 1:A and B – Strategy for the development of the live-cell marker and its binding to stable MTs within a cell. 1C shows the colocalizing of rigor-2xmNeongreen with acetylated stable MTs, near the perinuclear region of U2OS cells. (Adapted from Fig 1, Jansen et.al., 2021).

Next, the duration of rigor-2xmNeongreen binding with stable microtubules (labeled with rigor- 2xmNeongreen) was assessed in comparison to dynamic microtubules (labeled with mCherry-tubulin). TIRF(Total internal reflection fluorescence)-microscopy revealed that rigor-2xmNeongreen could be tracked on stable acetylated microtubules for at least thirty minutes, while dynamic microtubules (mCherry) show instability. Treatment with nocodazole (10uM), an MT destabilizing agent did not affect the bundle of rigor-2xmNeongreen stable MTs in U2OS cells. Further, serum starvation of Swiss 3T3 cells upon rigor expression, followed by 8h culture in serum added media, the rigor-2xmNeongreen MTs disassemble and resemble the stable MT network as in control cells.

Taken together, these experiments validate that rigor-2xmNeongreen could be used as a live-cell marker for stable MTs.

Rigor-2xmNeongreen can be used to study the dynamics of stable MTs:

Though stable microtubules are long-lived, they are dynamic and prone to mechanical forces. U2OS cells transfected with rigor-2xmNeongreen displayed distinct behaviors. Cytoplasmic microtubule transport, MT looping, undulation, and breakage were frequently observed in stable MTs. This indicates that stable microtubules may undergo fluctuations due to local pulling and pushing forces. The observation of such dynamic behaviors is restricted by immunocytochemistry on fixed cells, thus, demonstrating that rigor-2xmNeongreen may be a potential live-cell marker for exploring stable MTs dynamics.

A possible mechanism for the stability of stable microtubules using rigor-2xmNeongreen:

According to the authors, there are two possibilities for the stability of stable MTs: mechanisms that prevent its depolymerization, or the presence of stabilizing proteins/modifications along its length. To explore this, MTs expressing mCherry-tubulin and rigor-2xmNeongreen were photo-ablated in the perinuclear region of U2OS cells. Rigor-2xmNeongreen labeled MTs were stable on both sides of the cut. This suggests that the stability of stable MTs is uniform along the entire length. A detailed investigation is required to elucidate the exact mechanisms of the stability of the stable MTs.

Fig 2: A. Cartoon for the strategy of laser severing of microtubules. B. Yellow arrows marking rigor- 2xmNeongreen labeled microtubules retaining rigor expression on either side of the cut, time scale seconds: milliseconds. ( Adapted from Fig 7, Jansen et.al., 2021).

Fig 2: A. Cartoon for the strategy of laser severing of microtubules. B. Yellow arrows marking rigor- 2xmNeongreen labeled microtubules retaining rigor expression on either side of the cut, time scale seconds: milliseconds. ( Adapted from Fig 7, Jansen et.al., 2021).

Importance of this preprint:

This work is the first to develop a live cell marker (rigor-2xmNeongreen) for the direct visualization of stable microtubules. This tool can be an asset for the study of this microtubule subset in the future. Live cell tracking of stable microtubules may help to decipher cell organization, origin, types, and post-translational modifications. In addition, it may be feasible to elucidate the mechanisms of intracellular transport that remain largely unexplored.

Questions to the authors: 

  1. Are both stable and dynamic microtubules involved in cell division? If yes, can this marker be used to visualize and study stable microtubules during cell division?
  2. What are your thoughts on introducing this marker in model organisms(mouse/chick) to study microtubule organization and dynamics during early developmental processes?
  3. What do you speculate are the major differences between the surfaces of stable and dynamic MTs?

References

  1.  Gundersen, G.G. & Bulinski, J.C., 1988. Selective stabilization of microtubules oriented toward the direction of cell Proceedings of the National Academy of Sciences of the United States of America, 5(16), pp.5946– 5950.
  2. Cai, et al., 2009. Single molecule imaging reveals differences in microtubule track selection between Kinesin motors. PLoS Biology, 7(10), e1000216.
  3. Dunn, et al., 2008. Differential trafficking of Kif5c on tyrosinated and detyrosinated microtubules in live cells.Journal of Cell Science, 121(Pt 7), pp.1085–1095.
  4. Guardia, C.M. et al., 2016. BORC Functions Upstream of Kinesins 1 and 3 to Coordinate Regional Movement of Lysosomes along Different Microtubule Tracks. Cell reports, 17(8), 1950–1961.
  5. Rice, et al., 1999. A structural change in the kinesin motor protein that drives motility. Nature, 402(6763), pp.778– 784.
  6. Tas, P. et al., 2017. Differentiation between Oppositely Oriented Microtubules Controls Polarized Neuronal Transport. Neuron, 96(6), pp.1264–1271.e5
  7. Farías, G.G. et al., 2017. BORC/kinesin-1 ensemble drives polarized transport of lysosomes into the Proceedings of the National Academy of Sciences of the United States of America, 114(14), pp.E2955–E2964.
  8. Shaner, C. et al., 2013. A bright monomeric green fluorescent protein derived from Branchiostoma lanceolatum. Nature Methods, 10(5), pp.407–409.

Tags: dynamics, live-cell marker, microtubules

Posted on: 11th July 2021 , updated on: 12th July 2021

doi: https://doi.org/10.1242/prelights.29988

Read preprint (No Ratings Yet)




Have your say

Your email address will not be published. Required fields are marked *

This site uses Akismet to reduce spam. Learn how your comment data is processed.

Sign up to customise the site to your preferences and to receive alerts

Register here

preLists in the cell biology category:

Fibroblasts

The advances in fibroblast biology preList explores the recent discoveries and preprints of the fibroblast world. Get ready to immerse yourself with this list created for fibroblasts aficionados and lovers, and beyond. Here, my goal is to include preprints of fibroblast biology, heterogeneity, fate, extracellular matrix, behavior, topography, single-cell atlases, spatial transcriptomics, and their matrix!

 



List by Osvaldo Contreras

EMBL Synthetic Morphogenesis: From Gene Circuits to Tissue Architecture (2021)

A list of preprints mentioned at the #EESmorphoG virtual meeting in 2021.

 



List by Alex Eve

FENS 2020

A collection of preprints presented during the virtual meeting of the Federation of European Neuroscience Societies (FENS) in 2020

 



List by Ana Dorrego-Rivas

Planar Cell Polarity – PCP

This preList contains preprints about the latest findings on Planar Cell Polarity (PCP) in various model organisms at the molecular, cellular and tissue levels.

 



List by Ana Dorrego-Rivas

BioMalPar XVI: Biology and Pathology of the Malaria Parasite

[under construction] Preprints presented at the (fully virtual) EMBL BioMalPar XVI, 17-18 May 2020 #emblmalaria

 



List by Dey Lab, Samantha Seah

1

Cell Polarity

Recent research from the field of cell polarity is summarized in this list of preprints. It comprises of studies focusing on various forms of cell polarity ranging from epithelial polarity, planar cell polarity to front-to-rear polarity.

 



List by Yamini Ravichandran

TAGC 2020

Preprints recently presented at the virtual Allied Genetics Conference, April 22-26, 2020. #TAGC20

 



List by Maiko Kitaoka et al.

3D Gastruloids

A curated list of preprints related to Gastruloids (in vitro models of early development obtained by 3D aggregation of embryonic cells). Updated until July 2021.

 



List by Paul Gerald L. Sanchez and Stefano Vianello

ECFG15 – Fungal biology

Preprints presented at 15th European Conference on Fungal Genetics 17-20 February 2020 Rome

 



List by Hiral Shah

ASCB EMBO Annual Meeting 2019

A collection of preprints presented at the 2019 ASCB EMBO Meeting in Washington, DC (December 7-11)

 



List by Madhuja Samaddar et al.

EMBL Seeing is Believing – Imaging the Molecular Processes of Life

Preprints discussed at the 2019 edition of Seeing is Believing, at EMBL Heidelberg from the 9th-12th October 2019

 



List by Dey Lab

Autophagy

Preprints on autophagy and lysosomal degradation and its role in neurodegeneration and disease. Includes molecular mechanisms, upstream signalling and regulation as well as studies on pharmaceutical interventions to upregulate the process.

 



List by Sandra Malmgren Hill

Lung Disease and Regeneration

This preprint list compiles highlights from the field of lung biology.

 



List by Rob Hynds

Cellular metabolism

A curated list of preprints related to cellular metabolism at Biorxiv by Pablo Ranea Robles from the Prelights community. Special interest on lipid metabolism, peroxisomes and mitochondria.

 



List by Pablo Ranea Robles

BSCB/BSDB Annual Meeting 2019

Preprints presented at the BSCB/BSDB Annual Meeting 2019

 



List by Dey Lab

MitoList

This list of preprints is focused on work expanding our knowledge on mitochondria in any organism, tissue or cell type, from the normal biology to the pathology.

 



List by Sandra Franco Iborra

Biophysical Society Annual Meeting 2019

Few of the preprints that were discussed in the recent BPS annual meeting at Baltimore, USA

 



List by Joseph Jose Thottacherry

ASCB/EMBO Annual Meeting 2018

This list relates to preprints that were discussed at the recent ASCB conference.

 



List by Dey Lab, Amanda Haage

Also in the developmental biology category:

Fibroblasts

The advances in fibroblast biology preList explores the recent discoveries and preprints of the fibroblast world. Get ready to immerse yourself with this list created for fibroblasts aficionados and lovers, and beyond. Here, my goal is to include preprints of fibroblast biology, heterogeneity, fate, extracellular matrix, behavior, topography, single-cell atlases, spatial transcriptomics, and their matrix!

 



List by Osvaldo Contreras

EMBL Synthetic Morphogenesis: From Gene Circuits to Tissue Architecture (2021)

A list of preprints mentioned at the #EESmorphoG virtual meeting in 2021.

 



List by Alex Eve

EMBL Conference: From functional genomics to systems biology

Preprints presented at the virtual EMBL conference "from functional genomics and systems biology", 16-19 November 2020

 



List by Jesus Victorino

Single Cell Biology 2020

A list of preprints mentioned at the Wellcome Genome Campus Single Cell Biology 2020 meeting.

 



List by Alex Eve

Society for Developmental Biology 79th Annual Meeting

Preprints at SDB 2020

 



List by Irepan Salvador-Martinez, Martin Estermann

FENS 2020

A collection of preprints presented during the virtual meeting of the Federation of European Neuroscience Societies (FENS) in 2020

 



List by Ana Dorrego-Rivas

Planar Cell Polarity – PCP

This preList contains preprints about the latest findings on Planar Cell Polarity (PCP) in various model organisms at the molecular, cellular and tissue levels.

 



List by Ana Dorrego-Rivas

Cell Polarity

Recent research from the field of cell polarity is summarized in this list of preprints. It comprises of studies focusing on various forms of cell polarity ranging from epithelial polarity, planar cell polarity to front-to-rear polarity.

 



List by Yamini Ravichandran

TAGC 2020

Preprints recently presented at the virtual Allied Genetics Conference, April 22-26, 2020. #TAGC20

 



List by Maiko Kitaoka et al.

3D Gastruloids

A curated list of preprints related to Gastruloids (in vitro models of early development obtained by 3D aggregation of embryonic cells). Updated until July 2021.

 



List by Paul Gerald L. Sanchez and Stefano Vianello

ASCB EMBO Annual Meeting 2019

A collection of preprints presented at the 2019 ASCB EMBO Meeting in Washington, DC (December 7-11)

 



List by Madhuja Samaddar et al.

EDBC Alicante 2019

Preprints presented at the European Developmental Biology Congress (EDBC) in Alicante, October 23-26 2019.

 



List by Sergio Menchero et al.

EMBL Seeing is Believing – Imaging the Molecular Processes of Life

Preprints discussed at the 2019 edition of Seeing is Believing, at EMBL Heidelberg from the 9th-12th October 2019

 



List by Dey Lab

SDB 78th Annual Meeting 2019

A curation of the preprints presented at the SDB meeting in Boston, July 26-30 2019. The preList will be updated throughout the duration of the meeting.

 



List by Alex Eve

Lung Disease and Regeneration

This preprint list compiles highlights from the field of lung biology.

 



List by Rob Hynds

Young Embryologist Network Conference 2019

Preprints presented at the Young Embryologist Network 2019 conference, 13 May, The Francis Crick Institute, London

 



List by Alex Eve

Pattern formation during development

The aim of this preList is to integrate results about the mechanisms that govern patterning during development, from genes implicated in the processes to theoritical models of pattern formation in nature.

 



List by Alexa Sadier

BSCB/BSDB Annual Meeting 2019

Preprints presented at the BSCB/BSDB Annual Meeting 2019

 



List by Dey Lab

Zebrafish immunology

A compilation of cutting-edge research that uses the zebrafish as a model system to elucidate novel immunological mechanisms in health and disease.

 



List by Shikha Nayar
Close