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Inhibition of microtubule depolymerization by osmolytes George D. Bachand, Rishi Jain, Randy Ko, Nathan F. Bouxsein, and Virginia VanDelinder Biomacromolecules, Just Accepted Manuscript • DOI: 10.1021/acs.biomac.7b01799 • Publication Date (Web): 24 Apr 2018 Downloaded from http://pubs.acs.org on April 25, 2018

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Inhibition of microtubule depolymerization by osmolytes

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George D. Bachand,* Rishi Jain, Randy Ko, Nathan F. Bouxsein, and Virginia VanDelinder

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Center for Integrated Nanotechnologies, Sandia National Laboratories, PO Box 5800, MS 1303, Albu-

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querque, NM 87185

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Keywords: tubulin, osmotic pressure, biopolymer, polyethylene glycol, trimethylamine-N-oxide, dy-

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namic instability

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ACS Paragon Plus Environment

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ABSTRACT

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Microtubule dynamics play a critical role in the normal physiology of eukaryotic cells, as well as a

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number of cancers and neurodegenerative disorders. The polymerization/depolymerization of microtu-

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bules is regulated by a variety of stabilizing and destabilizing factors, including microtubule-associated

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proteins and therapeutic agents (e.g., paclitaxel, nocodazole). Here we describe the ability of the osmo-

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lytes polyethylene glycol (PEG) and trimethylamine-N-oxide (TMAO) to inhibit the depolymerization

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of individual microtubule filaments for extended periods of time (up to 30 days). We further show that

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PEG stabilizes microtubules against both temperature- and calcium-induced depolymerization. Our re-

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sults collectively suggest that the observed inhibition may be related to combination of the kosmotropic

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behavior and excluded volume/osmotic pressure effects associated with PEG and TMAO. Taken togeth-

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er with prior studies, our data suggest that the physiochemical properties of the local environment can

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regulate microtubule depolymerization, and may potentially play an important role in in vivo microtu-

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bule dynamics.

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INTRODUCTION

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Microtubules are one of three polymer filaments that compose the cytoskeleton of eukaryotic cells.

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They play a critical role in metabolic processes such as chromosome segregation during cell division,

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but are also fundamental to the pathologies of cancer and neurodegenerative diseases (e.g., Alzheimer’s

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and Parkinson’s disease).1–5 A unique property of microtubules is their ability to stochastically switch

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between states of growth and catastrophic shrinkage, a phenomenon known as dynamic instability.6

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During polymerization, αβ tubulin dimers associate longitudinally to form polar protofilaments, which

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subsequently interact laterally to form a sheet that closes into a mature tubular filament. Tubulin dimers

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possess two nucleotide binding sites: (i) the non-exchangeable (N) site in α tubulin that is constitutively

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occupied by guanosine triphosphate (GTP), and (ii) the exchangeable (E) site on β tubulin that hydro2


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lyzes GTP during growth.7 Hydrolysis of GTP induces compaction around the E-site, causing an ener-

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getically unfavorable, bent tubulin structure.8 This strain does not affect growth or stability as long as

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there is GTP present in αβ tubulin dimers at the end of the microtubules (i.e., GTP cap). If the GTP cap

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is lost, the strain is believed to initiate peeling of the protofilaments, generating a “ram’s horns” mor-

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phology9 and catastrophic depolymerization of the microtubule.7,10

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The polymerization and organization of cytoskeletal filaments, including microtubules, are strongly

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influenced by the physiochemical nature of the solvent. For example, both actin and microtubule fila-

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ments readily align and aggregate in crowded environments due to the osmotic forces and torque associ-

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ated with the background macromolecules.11–13 Here, macromolecular crowding produce excluded vol-

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ume effects and elevate the osmotic pressure,14–16 as well as changes in viscosity, diffusion, weak/soft

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interaction, and solvation.17–19 Increased rates of actin and tubulin polymerization have also been report-

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ed in presence of osmolytes and in crowded environments.20–23 Wieczorek et al. reported that the rate of

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tubulin polymerization approached its theoretical limit with the addition of large macromolecules (e.g.,

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polyethylene glycol (PEG), bovine serum albumin (BSA)), but not small solutes (e.g., glycol, ethylene

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glycol).23 Similarly, Schummel et al. observed an enhancement in the rate of tubulin polymerization us-

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ing Ficoll as a macromolecular crowding agent.25 In the same study, the addition of the osmolyte trime-

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thylamine-N-oxide (TMAO) was shown to modulate the polymerization of tubulin and offset the inhibi-

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tory effect of urea.25 This observation was consistent with prior reports in which TMAO, but not other

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methylamines (e.g., betaine, glycine), stimulated the polymerization of tubulin.24,25 This effect is likely

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related to the known preferential exclusion of TMAO from protein surfaces and enhancement of the hy-

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drogen-bonding network in the surrounding water.26,27

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In this study, we describe the inhibition of microtubule depolymerization by the osmolytes using fluo-

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rescence microscopy. Here, the addition of PEG and TMAO to solutions of microtubules results in an

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inhibition of depolymerization that persists for at least 30 days. In contrast, this inhibition was not ob-

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served in the presence of sucrose, ethylene glycol (EG), glycerol, polyvinylpyrrolidone (PVP), or BSA. 3



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We further show that PEG600 significantly inhibits both the calcium- and temperature-induced depoly-

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merization of microtubules. Collectively, our data provides new insights on the role of physiochemistry

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in microtubule depolymerization and broadly expands our understanding of microtubule dynamics.

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EXPERIMENTAL SECTION

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Chemicals and Buffers. All chemicals were purchased from Sigma-Aldrich unless indicated other-

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wise. BRB80 buffer contained 80 mM PIPES, 1 mM MgCl2, and 1 mM EGTA, and was adjusted to a

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pH value of 6.9 with KOH. GPEM buffer was prepared by adding 1 mM GTP and 10% glycerol to

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BRB80 buffer. BRB80T buffer contained BRB80 with 10 µM paclitaxel (Taxol®). BRB80CA buffer

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was prepared by adding 0.2 mg mL-1 casein and 1 mM AMP-PNP to BRB80. Imaging solution was pre-

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pared with by adding 0.2 mg mL-1 casein, 1 mM β,γ-imidoadenosine 5′-triphosphate (AMP-PMP, a non-

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hydrolyzable analog of ATP), 10 mM paclitaxel, and an anti-fade mix that included 0.02 mg mL-1 glu-

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cose oxidase, 0.008 mg/mL catalase, 20 mM D-glucose, 1 mM 6-hydroxy-2,5,7,8-tetramethylchroman-

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2-carboxylic acid, and 1 mM DTT to BRB80 buffer.

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Polyethylene glycol 600 (PEG600) solutions were prepared by combining PEG600 with BRB80 in a

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given weight/volume (w/v) ratio, e.g. 2 g PEG600 in 10 ml BRB80 is 20% w/v. BRB80-PEG imaging

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solutions contained 0.2 mg mL-1 casein, 1 mM AMP-PNP, 0.02 mg mL-1 glucose oxidase, 0.008 mg

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mL-1 catalase, 20 mM D-glucose, 1 mM trolox, and 1 mM DTT. Solutions of additional osmolytes (25%

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w/v) including sucrose, EG, TMAO, polyvinylpyrrolidone (PVP), bovine serum albumin (BSA), and

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glycerol were also prepared in BRB80 as described above.

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MT Preparation. Lyophilized porcine brain tubulin (Cytoskeleton, Inc.) consisting of Hilyte™ Fluor

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488 fluorescently labelled tubulin and unlabeled tubulin in a 1:3 molar ratio was suspended in GPEM

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buffer at a concentration of 22 mM and then polymerized for 30 minutes at 37°C. After polymerization,

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the microtubules for control experiments were diluted to 0.5 mM in BRB80T to prevent depolymeriza-

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tion. For the majority of experiments, microtubules were stabilized with PEG600 solutions ranging from 4


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5 to 32% (w/v). For all other osmolytes, the stability of microtubules was evaluated in 25% (w/v) solu-

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tion prepared in BRB80. All samples were stored at room temperature unless specifically denoted.

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Viscosity and Osmotic Pressure. The viscosities of aqueous polymer solutions were measured using

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a Rheometric Scientific RFS3 strain-controlled rheometer. Each measurement used 15 mL of polymer

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solution loaded into a Couette fixture with a 34-mm diameter cup and a 32-mm diameter bob. Viscosity

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was measured as a function of shear rate from 0.1 to 1000 s-1 at a temperature of 20°C. All measured

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samples exhibited Newtonian behavior. The reported viscosities are averaged over a range of shear rates

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from 10 to 100 s-1, which produced sufficiently measurable torque in all samples. Osmotic pressure val-

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ues for PEG600, PEG10K, PVP10K, BSA, and TMAO were determined based on published data.28–30

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For sucrose, glycerol, and EG were calculated by ߨ = Mܴܶ, where M is the molarity of the solution, R

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is the ideal constant, and T is the temperature in °K.

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Glass flow cell preparation. A flow cell was created using a glass slide and coverslip adhered by two

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strips of double-sided tape, resulting in a 5 mm x 20 mm x 0.15 mm channel. The motor protein KIF5B

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(here on called kinesin) was used to immobilize MTs to the coverslip surface for imaging. A non-

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hydrolysable ATP analog, AMP-PNP, was used to prevent motor function. The expression and purifica-

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tion of the kinesin has been reported previously.31 Initially, the channel was filled with 1 mM kinesin in

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BRB80CA. After a 5-min incubation, 20 mL of 0.5 mg mL-1 MTs in either plain or PEG imaging solu-

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tion was introduced into the channel. After another 5-min incubation, the channel was washed with ei-

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ther the PEG or the plain imaging solution. Valant sealant was used to close both sides of the channel to

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prevent drying. Immediately afterwards, the channel was placed on the microscope for observation.

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Microscope and Data Analysis. Fluorescence characterization was performed on an IX-81 Olympus

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microscope with either a 60X/1.42 NA or 100X/1.4 NA oil immersion objective, Semrock Brightline

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Pinkel DA/FI/TR/Cy5/Cy7-5X-A000 filter set, 25 ND filter, and Orca Flash 4.0 digital camera. For lon-

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gevity experiments, microtubules were characterized by fluorescence microscopy on days 0, 1, 5, 14,

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and 30 post-polymerization and stabilization. For images in which depolymerization was quantitatively 5



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assessed, images were acquired at 20 s intervals for a total duration of 30 min. The endpoints of the MT

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filaments were tracked in ImageJ to measure disassembly rates with no post-processing performed on

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the recorded images.

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Subtilisin digestion of tubulin C-terminal tails. Paclitaxel-stabilized MTs were incubated in 100 µg

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mL-1 of subtilisin for 60 min at 37 °C to remove the tubulin C-terminal tails.32 The digestion was halted

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by adding 5 mM phenylmethylsolfonyl fluoride (PMSF), and then the solution was placed on top of a

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40% glycerol cushion (BRB80, 10 µM taxol, 40% glycerol) and centrifuged at 190,000 x g for 25 min.

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The supernatant was discarded and the pelleted microtubules were re-suspended in BRB80T. The diges-

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tion was confirmed on an SDS-page gel.

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PEG Entry into MT lumen. After polymerization, microtubules stabilized with BRB80T were incu-

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bated with 80 µM mPEG-rhodamine 550 (Creative PEG Works) for 1 week. A glass flow cell was filled

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with 1 mM kinesin in BRB80CA and allowed to incubate for 5 min. Microtubules diluted 5 µM in either

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PEG motility solution with 80 µM TRITC-labelled PEG were then introduced to the channel. After 5

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min, the channel was washed with either plain or PEG imaging solution and then imaged immediately.

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RESULTS AND DISCUSSION

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PEG-induced stabilization of microtubule filaments. Depolymerization of PEG-stabilized microtu-

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bules (25% PEG600) was inhibited over the course of 30 days, similar to that observed with paclitaxel,

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a known microtubule-stabilizing agent (Figure 1A). Both the average length and number of microtu-

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bules per mm2 varied over time for microtubules stabilized with PEG and paclitaxel (Figure 3b, c) (p
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High

Glycerol

92.09

2.71

2.1

6.6

Intermediate

Sucrose

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0.73

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Low

BSA

66,500

0.004

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0.07

Low

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Figure 2. (a) Fluorescence photomicrographs of microtubules acquired at t = 0, 1, 8, and 24 h after polymerization and addition of 25% w/v solution of different osmolytes dissolved in BRB80. Images were not acquired after t = 0 h for BRB80, BSA, and sucrose as no microtubules were present. Scale bars = 20 µm.

Interactions of PEG600 with microtubules. It is possible that the observed inhibition of depolymerization may be related to direct interaction between PEG and the outer or inner surfaces of tubulin. In particular, the C-terminal tails (CTTs) of tubulin are known to play important role in microtubule dynamics,39,40 and thus are a potential domain that may interact with PEG. To test this hypothesis, microtubules lacking CTTs were prepared stabilized paclitaxel, digested with subtilisin A (Figure S3), and tested with respect to stabilization with 25% PEG600. Here, microtubules displayed similar stability 9


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(i.e., >30 min) with paclitaxel and PEG600, regardless of the presence/absence of CTTs (Figure 3). In contrast, the majority of microtubules with and without CTTs depolymerized within one minute following the introduction of BRB80 (Figure 3). Thus, these data suggest that that the enhancement in stability is not based on direct interactions on the CTTs.

Figure 3. Fluorescence photomicrographs of microtubules without (top) and with (bottom) C-terminal tails (CTTs) under various buffer conditions. Scale bar = 20 µm

Diffusion of PEG into the lumen of microtubules has previously been shown,13 raising the question as to whether PEG interacts with the interior surface of the microtubule. The PEG600 used in our experiment has an Rg of ~1 nm,41 and thus should readily diffuse into the lumen of the microtubule filament.42 To further assess this issue, a fluorescently-labeled PEG (mPEG-Rhodamine, 550 Da molecular weight) was used to visualize whether PEG is able enter the lumen. Microtubules were stabilized with 25% mPEG-Rhodamine for one week to ensure sufficient time for diffusion and entry into the lumen. Microtubules were then introduced into a kinesin-coated flow cell, and subsequently washed with 25% PEG600. If the fluorescent PEG entered the lumen, co-localization of green (tubulin) and red (mPEGRhodamine) fluorescence would have been expected initially, followed by the disappearance of the red fluorescence over time as the fluorescent PEG diffuses out of the lumen from the microtubule ends. No co-localization of fluorescence was observed in these experiments (Figure S3), suggesting that PEG 10


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does not enter or interact with the lumen of the microtubule. A caveat of this experiments is that the microtubule lattice has lateral pores (~2 nm2),43 in addition to their ends, through which mPEG-Rhodamine could have rapidly diffused. If the diffusion of PEG through lateral pores in the microtubule lattice was fast enough, our experiments would not have detected the presence of PEG inside the lumen on the microtubules. It is also possible that fluorescent PEG was present at a concentration below that detectable by fluorescence microscopy. As such, further characterization (e.g., fluorescence correlation spectroscopy) is necessary to provide a more conclusive understanding with respect to interactions between PEG and the inner and outer surfaces of the microtubule. PEG600 stabilization against temperature- and calcium-induced depolymerization. Aside from their intrinsic dynamic instability, depolymerization of microtubules is influenced by a wide range of factors including temperature,44–46 presence/absence of microtubule-associated proteins (e.g., tau, MCAK)47, and metal cations.48,49 Cold temperature-induced depolymerization, for example, is commonly used in the purification of tubulin,50 as well as to characterize microtubule stabilizing agents.51–53 In the present study, we examined the ability of PEG to stabilize microtubules against both temperatureinduced depolymerization by incubating microtubules stabilized with either paclitaxel or PEG600 at a specific temperature for 24 h, and characterizing their presence/absence with fluorescence microscopy. The addition of 32% and 25% PEG600 stabilized microtubules against depolymerization (Figure 4 and S5, respectively), extending the temperature stability range (-20 to 45°C) in comparison to those stabilized with paclitaxel (15 to 35°C). This effect was more prominent at lower temperatures where depolymerization results from entropically driven processes54 rather than thermal denaturation of tubulin.48 Similar results were observed using microtubules that were polymerized with guanosine-5'-[(α,β)methyleno]triphosphate (GMPCPP), a non-hydrolyzable GTP analog (Figure S5), which allows the tubulin and protofilaments to maintains straight, “GTP-like” con-formation.8 GMPCPP-polymerized microtubules stabilized with PEG600 displayed enhanced stability at lower temperatures, but not at elevated temperature when compared to GTP-polymerized microtubules stabilized with PEG600. Overall 11


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these data suggest that the PEG600 can inhibit the depolymerization of microtubules whether tubulin dimers are either in the straight conformation (GMPCPP), or the bent conformation associated with the hydrolysis of GTP to GDP.

Figure 4. The addition of 32% PEG600 induces an osmotic pressure (~4.4 MPa) sufficient to prevent the temperatureinduced polymerization of microtubules ( 0.05; Figure S6), an effect remained constant even in the presence of 5 mM Ca2+ (Figure 5a, b). A strong correlation between the osmotic pressure induced by PEG600 and the percent inhibition in depolymerization in the presence of Ca2+ (Figure 5c), which was calculated using the rates of depolymeri-

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zation compared against that observed in the presence of 5% PEG600 (i.e., depolymerization rate at given percent PEG600

Figure 5. Microtubule depolymerization rates at the (a) minus ends in the presence () or absence () of Ca2+, and (b) plus ends in the presence () or absence () of Ca2+. Error bars = standard error of the mean derived from linear regression. (c) Inhibition of Ca2+-induced depolymerization of the minus () and plus () ends of the microtubule as a function of the PEG concentration. Inhibition was calculated by dividing the rate of depolymerization at a given concentration by that observed for 5% PEG600. Error bars= propagated uncertainties.

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/ depolymerization rate with 5% PEG600). The inhibition of microtubule depolymerization reached an apparent maximum at ~20% PEG600 (Figure 5c). The non-linear relationship between PEG600 and inhibition of microtubule depolymerization is consistent with similar effects reported for enzyme activity55,56 and RNA folding/unfolding thermodynamics57 in crowded environments.

CONCLUSIONS

In summary, we have shown that the osmolytes PEG and TMAO inhibit the depolymerization of microtubules in ex vivo environments. This inhibition may be attributed to the at least two aspects: (i) the kosmotropic behavior of these osmolytes, and (ii) osmotic pressure/excluded volume effects. Taken together with the reported enhanced polymerization rates of tubulin,23,24,27 a number of fundamental questions may be raised in terms of the roles of macromolecular crowding, protein solvation, osmotic pressure, and excluded volume effects on the ex vivo and in vivo dynamics of microtubule filaments. Osmolytes such as TMAO can regulate the macromolecular crowding and water content of cells, helping to maintain a balanced level of crowding and avoid detrimental effects due osmotic stresses.58 Modulation of the free volume can also have significant effects on weak interactions among proteins and potentially serve as a means of regulating functions inside of a cell.59 Interestingly, microtubules has been shown to mechanically slow down the initial swelling and volume regulation of cells following an osmotic stress.60 This relationship, along with the results our study, provide an impetus for developing a deeper understanding of the relationship between osmolytes and in vivo microtubule dynamics.

ASSOCIATED CONTENT

Supporting Information The Supporting Information is available free of charge on the ACS Publications website at DOI: Additional experimental data characterizing the bundling and stability of microtubules, cleavage of the C-

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terminal tails, presence of fluorescent PEG in microtubules, temperature and calcium-induced depolymerization, and detailed statistical analyses.

AUTHOR INFORMATION

Corresponding Author *E-mail: [email protected]

ORCID George D. Bachand: 0000-0002-3169-9980

Author Contributions R.J. and N.F.B. initially observed the PEG stabilization phenomenon. G.D.B., N.F.B, and V.V. developed research plan. All authors contributed to the design of specific experiments. N.F.B. and R.J. performed stabilization and bundling experiments. R.J. and R.K. performed temperature experiments. R.K. performed long-term stability, alternate osmolytes, and calcium-dependent depolymerization experiments. V.V. and R.K. performed C-terminal tails experiments. V.V. performed fluorescent PEG experiments. V.V. performed image analysis of long-term stability experiments. G.D.B. performed statistical analyses, and wrote the manuscript with input from all authors.

Funding Sources U.S. Department of Energy, Office of Basic Energy Sciences, Division of Materials Sciences and Engineering

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ACKNOWLEDGMENTS

We sincerely thank Dr. Mark Stevens for his critical review, and Dr. Brad Jones for performing viscosity measurements and insightful discussion regarding the results presented in this paper. This work was supported by the U.S. Department of Energy, Office of Basic Energy Sciences, Division of Materials Sciences and Engineering (BES-MSE). This work was performed, in part, at the Center for Integrated Nanotechnologies, an Office of Science User Facility operated for the U.S. Department of Energy (DOE) Office of Science (user project number RA2015A0004). Sandia National Laboratories is a multimission laboratory managed and operated by National Technology & Engineering Solutions of Sandia, LLC, a wholly owned subsidiary of Honeywell International Inc., for the U.S. Department of Energy’s National Nuclear Security Administration under contract DE-NA0003525. The views expressed in the article do not necessarily represent the views of the U.S. Department of Energy or the United States Government.

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Figure 1. (a) Fluorescence photomicrographs of microtubule stabilized with PEG600 or paclitaxel at various time intervals over the course of 30 days. (b) Number of microtubules per mm2 and (c) microtubule length over time for samples stabilized with PEG600 (ν) or paclitaxel (ν). Error bars = standard deviation. Scale bars = 15 µm. 127x106mm (300 x 300 DPI)



Figure 2. (a) Fluorescence photomicrographs of microtubules acquired at t = 0, 1, 8, and 24 h after polymerization and addition of 25% w/v solution of different osmolytes dissolved in BRB80. Images were not acquired after t = 0 h for BRB80, BSA, and sucrose as no microtubules were present. Scale bars = 20 µm. 76x35mm (300 x 300 DPI)


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Figure 3. Fluorescence photomicrographs of microtubules without (top) and with (bottom) C-terminal tails (CTTs) under various buffer conditions. Scale bar = 20 µm. 55x37mm (300 x 300 DPI)



Figure 4. The addition of 32% PEG600 induces an osmotic pressure (~4.4 MPa) sufficient to prevent the temperature-induced polymerization of microtubules (

Inhibition of Microtubule Depolymerization by ... - ACS Publications

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