Speed effects on gliding motility assays due to surface

Speed effects on gliding motility assays due to surface passivation, water isotope, and osmotic stress.

Andy Maloney, Larry Herskowitz, Steve KochUniversity of New Mexico"Kiney"

This work is supported by the DTRA CB Basic Research Program under Grant No. HDTRA1-09-1-008 and the UNM IGERT on Integrating Nanotechnology with Cell Biology and Neuroscience NSF Grant DGE-0549500.We would also like to thank Susan Atlas, PI of the DTRA project and Dr. Haiqing Liu for supplying us kinesin-1.

Open Notebook Science

Surface passivation effects Water isotope effects Osmotic stress effects

MotivationWater plays an important role in biological systems and it is often overlooked4. It is a key component for the hydrolysis of ATP and has a crucial role in how proteins interact with one another due to the hydration of their surfaces. To the best of our knowledge, no work has been done on investigating how the kinesin-1 microtubule system changes when changes with water isotopes occur. Changing the water isotopes from hydrogen to deuterium or 16O to 18O may affect how kinesin interacts with microtubules. This change can be used as a novel experimental knob to probe features of the kinesin and microtubule system in new and different ways.

ExperimentAll chemicals were purchased from Sigma unless noted otherwise. Slide preparation was performed as follows:

• 10 minute incubation of slide with 0.5mg/mL α casein in PEM.• 5 minute incubation of slide with 0.25mg/mL α casein, 30µg/mL

kinesin-1, and 1mM ATP in PEM.• Introduction of Taxol stabilized microtubules with an enzymatic

antifade system and 1mM ATP in PEM with the desired mix of water isotopes. After sealing, observations are conducted immediately.

When using D2O, care must be taken in order to measure the correct "pH" denoted as the pD = pH + 0.415. Also, all solutions that have Taxol in them are always prepared fresh and are discarded after 3 hours to prevent Taxol crystals from forming6.

References1. Verma, Vivek, William Hancock, and Jeffrey Catchmark. “The role of casein in supporting the operation of surface bound kinesin.” Journal of Biological Engineering 2, no. 1 (2008): 14.2. Fox P, McSweeney P. Chapter 4: Milk Proteins. In: Dairy chemistry and biochemistry. 1 ed. London: Blackie Academic & Professional; 1998:146-238.3. Fox P, McSweeney P. Chapter 9: Heat-induced changes in milk. In: Dairy Chemistry and Biochemistry. 1 ed. London: Blackie Academic & Professional; 1998:347-378.4. Parsegian, V A, R P Rand, and D C Rau. 1995. Macromolecules and water: probing with osmotic stress. Methods in enzymology 259.5. Covington, Arthur K., Maya Paabo, Robert Anthony Robinson, and Roger G. Bates. 1968. Use of the glass electrode in deuterium oxide and the relation between the standardized pD (paD) scale and the operational pH in heavy water. Analytical Chemistry 40, no. 4: 700-706.6. Foss, Margit, Buck W L Wilcox, G Bradley Alsop, and Dahong Zhang. 2008. Taxol crystals can masquerade as stabilized microtubules. PloS one 3, no. 1: e1476.7. Böhm, K J, P Steinmetzer, a Daniel, M Baum, W Vater, and E Unger. 1997. Kinesin-driven microtubule motility in the presence of alkaline-earth metal ions: indication for a calcium ion-dependent motility. Cell motility and the cytoskeleton 37, no. 3: 226-31.8. Chase, P B, Y Chen, K L Kulin, and T L Daniel. 2000. Viscosity and solute dependence of F-actin translocation by rabbit skeletal heavy meromyosin. American journal of physiology. Cell physiology 278, no. 6: C1088-98.9. Highsmith, S, K Duignan, R Cooke, and J Cohen. 1996. Osmotic pressure probe of actin-myosin hydration changes during ATP hydrolysis. Biophysical journal 70, no. 6: 2830-7.10.Chaen, S. 2001. Effect of deuterium oxide on actomyosin motility in vitro. Biochimica et Biophysica Acta (BBA) - Bioenergetics 1506, no. 3: 218-223.11.Chaen, Shigeru, Naoto Yamamoto, Ibuki Shirakawa, and Haruo Sugi. 2003. In vitro actomyosin motility in deuterium oxide. Advances in experimental medicine and biology 538: 183-92; discussion 192.

!"#$%&'(")(*+"'",+-&'(.%,+%//$+%,!"##$%!!&'( )**+&,,---./0123456.276,825*45*,",','(

9:64!;!2<!'#

01&,/(%#23/$(%"4()"$(-+4&4+"%(1#$1"5/56

by fluorescence microscopy. Glass coverslips were coatedwith rhodamine labeled whole, !, " and # casein, washedin an antifade solution containing BRB80, 20 mM D-glu-cose, 20 $g/ml glucose oxidase, 8 $g/ml catalase and 0.5%"-merceptoethanol to reduce rhodamine bleaching, andvisualized using epi-fluorescence microscopy. A secondset of glass cover slips were first coated with unlabeledcasein, then exposed to fluorescently-labeled casein, andfinally antifade solution was washed in. The intensity ofthe rhodamine fluorescence on the surface was quantifiedfor each case and the intensity corrected for the back-ground signal in the absence of any fluorescent label. Datafor the two conditions are presented in figure 4. Rhodam-ine labeling extent of casein and its subunits was takeninto consideration. UV spectrophotometer analysis wasperformed on rhodamine labeled caseins and the fluores-

cence intensity was normalized accordingly. When rhod-amine labeled whole casein or casein subunits wasexposed to the clean glass surface, a tightly bound layer ofcasein adsorbed to the surface that was not washed awayby replacing the protein solution with the antifade solu-tion. However, when unlabeled casein was first intro-duced into the flow cell followed by the labeled casein,the measured intensity decreased in all cases. In the caseof whole casein and " casein, this decrease was substan-tial, representing a reduction of 48% and 34%, respec-tively. These data suggest that a bi-layer structure isformed for the whole casein and " casein, and that the sec-ond layer is more loosely bound. The very small differencein the measured intensities for # casein again suggests adifference in how this casein interacts with the glass sur-face and kinesin and provides no evidence for formation

Comparing the effectiveness of different casein subunits on kinesin functionFigure 3Comparing the effectiveness of different casein subunits on kinesin function. (a) The average number of microtu-bules was observed in a standard motility assay at low (0.8 $g/ml) and high (8 $g/ml) motor concentrations where casein was included in the surface blocking, kinesin adsorption and microtubule motility steps. For different casein subunits, (!, " and #) all solutions contained only that specific subunit. (b) Screen shots showing microtubule density on glass surface for different casein and high kinesin. Microtubules less than 1 $m were not counted. Scale bar is 10 $m.

!"#

0

10

20

30

40

50

60

70

Whole Alpha Beta Kappa

Nu

mb

er

of

Mic

rotu

bu

les

Kinesin 0.8ug/ml Kinesin 8ug/ml

$"#

## %&'()#*!+),-#!-.#

&,/&#0,-)+,-##*!+),-#!-.#&,/&#0,-)+,-#

#*!+),-#!-.#&,/&#0,-)+,-#

##*!+),-#!-.#&,/&#0,-)+,-#

#

Whole casein1

10 µm

!"#$%&'(")(*+"'",+-&'(.%,+%//$+%,!"##$%!!&'( )**+&,,---./0123456.276,825*45*,",','(

9:64!;!2<!'#

01&,/(%#23/$(%"4()"$(-+4&4+"%(1#$1"5/56




!"#

0

10

20

30

40

50

60

70


Nu

mb

er

of

Mic

rotu

bu

les


$"#

## %&'()#*!+),-#!-.#

&,/&#0,-)+,-##*!+),-#!-.#&,/&#0,-)+,-#

#*!+),-#!-.#&,/&#0,-)+,-#

##*!+),-#!-.#&,/&#0,-)+,-#

#

Alpha casein1

10 µm

!"#$%&'(")(*+"'",+-&'(.%,+%//$+%,!"##$%!!&'( )**+&,,---./0123456.276,825*45*,",','(

9:64!;!2<!'#

01&,/(%#23/$(%"4()"$(-+4&4+"%(1#$1"5/56




!"#

0

10

20

30

40

50

60

70


Nu

mb

er

of

Mic

rotu

bu

les


$"#

## %&'()#*!+),-#!-.#

&,/&#0,-)+,-##*!+),-#!-.#&,/&#0,-)+,-#

#*!+),-#!-.#&,/&#0,-)+,-#

##*!+),-#!-.#&,/&#0,-)+,-#

#

Beta casein1

10 µm

!"#$%&'(")(*+"'",+-&'(.%,+%//$+%,!"##$%!!&'( )**+&,,---./0123456.276,825*45*,",','(

9:64!;!2<!'#

01&,/(%#23/$(%"4()"$(-+4&4+"%(1#$1"5/56




!"#

0

10

20

30

40

50

60

70


Nu

mb

er

of

Mic

rotu

bu

les


$"#

## %&'()#*!+),-#!-.#

&,/&#0,-)+,-##*!+),-#!-.#&,/&#0,-)+,-#

#*!+),-#!-.#&,/&#0,-)+,-#

##*!+),-#!-.#&,/&#0,-)+,-#

#

Kappa casein1

10 µm

Very nice work done by Verma et. al.1 (above) shows that microtubules are supported differently in the gliding motility assay dependent on the type of casein used as a passivator. Building on what they have done (below), we investigated the speed changes microtubules glide at depending on what type of passivation is used. Our preliminary work notes that kappa casein is by far the worst at supporting the gliding motility assay at the kinesin concentration we use, 30µg/mL. Images are false colored using ImageJ.

MotivationDuring the stepping process, the kinesin motor domains bind and unbind from their binding sites on the microtubules. Binding and unbinding rates of biomolecules are highly dependent on hydration and exclusion of water from the binding interface. Osmotic stress will likely strongly affect the binding and unbinding rates for kinesin and thus may offer a tool to specifically probe those steps.

Experiment• All chemicals were purchased from Sigma unless noted otherwise.

Slide preparation was done exactly as stated in the Experiment part of Water isotope effects. We chose to use α casein as the surface passivator since it showed the closest speed similarities to whole casein which is the passivator most people use.

• A stock solution of 5M Betaine (Sigma-61962) in PEM was prepared and diluted into the motility solution for the experiments.

• Future work will include how motility is affected with the addition of other known osmolytes: Sarcosine (Sigma-S7672), D-Proline (Sigma-858919), TMAO (Sigma-317594), and other chemicals.

• In order to determine if osmotic stress is the key descriptor as to why gliding speeds are changed, viscosity measurements must be done on the assay solutions. Betaine is known to have less increase in viscosity than sucrose for example, however, viscosity remains a confounding issue that we need to explore.

Data analysisMicrotubules are tracked using custom LabVIEW software. Please see Larry Herskowitz's poster for a more detailed explanation of the tracking software. Briefly, data is taken from a total of 15 regions in each slide. Data is smoothed using a sliding Gaussian window to eliminate Brownian noise. We then determine the probability desinty function for instantaneous microtubule speed from a kernel density estimation using a Gaussian kernel of width 50nm/s. The peak of the PDF is used as the speed for that ROI. An interesting feature of the data is that the microtubules speed up overtime in all assays. The β casein passivation supports the fastest microtubule gliding speed while the κ casein passivation is the worst at supporting the gliding assay for these conditions. Each ROI, and correspondingly each data point, is illuminated for approximately 2 minutes. The illumination source is a 100W Hg lamp that is attenuated by 94% and sent through a standard TRITC filter cube.

Results• The tracking software is capable of

tracking both the positive end (unfilled markers) and the minus end (filled markers) of the microtubule. Plus and minus ends are offset for clarity in the plot. We use the two tracked numbers as a check to ensure that the microtubules are tracked properly. See Larry Herskowitz's poster for a more detailed description of the tracking software.

• H218O (Sigma-329878, black markers, 97% 18O) is quite expensive. We

purchase this water 1 gram at a time. In order to prepare the PEM solution with H2

18O water, a 10x solution of PEM is prepared in regular H2O. It is then diluted by a factor of 10 in H2

18O. What is plotted above is the % concentration of H2

18O used in an assay and not the % concentration of 18O in solution.

• While time prevented us from completing the experiment, You can clearly see the reduced speed trend by increasing H2

18O concentration in the assay.

• D2O (Sigma-151882, red markers, 99.9% D) is not as expensive as H218O

and so PEM is prepared in D2O. Again, what is plotted is the % D2O used in the assay and not the % deuterium in solution.

• A distinctive trend is observed as one reduces the amount of H2O in the assay.

• Data points at 70% and 80% D2O may be an experimental error and thus may not represent the true nature of the system.

• Error bars are the SEM of 8 ROIs where the slight increase in speed over the 8 regions is ignored (see data in the surface passivation section).

9% H218O

20 µm 20 µm

18% H218O

20 µm

81% H218O

10% D2O

20 µm 20 µm

50% D2O 90% D2O

20 µm

Results• Even the 0mM Betaine data set has a

significant osmotic pressure relative to regular water due to the osmotic agents present in the standard motility solution.

• The antifade system constitutes a portion of the initial osmotic pressure since it contains glucose oxidase (Sigma-G6641), catalase (Sigma-C9322), BME (Sigma-63689), and 20mM glucose (Sigma-49139).

• Initial studies show that gliding speed is affected by osmotic stress or viscosity. We are still unsure as to which of these is the strongest affect. Studies8-11 have been done on myosin in osmotic or viscous stress, however, no consensus has been made as to what the actual affect is coming from.

700

600

500

400

300

200

Spee

d (n

m/s

)

25002000150010005000

Time (s)

0mM Betaine Ø osmotic pressure1000mM Betaine2000mM Betaine

DiscussionWhen preparing the samples for the betaine tests, an interesting observation was made with the slide preparations. As can be seen in the images to the left, microtubules for the most part are straight, except for the wiggles which we believe is due to the osmotic stress affect. When preparing these slides, no casein was included in the motility solution that contained the microtubules.

When casein was added to the motility solution, odd squiggle patterns were seen in the assay. This observation led us to not include casein in our motility assay despite literature recommendations1. Casein was added in the motility solution for the surface passivation studies since those assays were performed before this observation was made.

AcknowledgementsWe would like to thank the following people for their extensive help in this project:

• Dr. Haiqing Liu at CINT for supplying the kinesin-1.• Dr. Susan Atlas PI of the DTRA project.• OpenWetWare for hosting Open Notebook Science.• LabVIEW for continued support.• Anthony Salvagno for insight with the cartoons.

MotivationFor kinesin to function properly in a gliding motility assay, the substrate to which kinesin adheres must first be passivated1. Functionalization of glass slides and slips is typically performed with bovine milk proteins called caseins, however, BSA can also be used as a passivator7. Bovine casein is a globular protein that can be broken up into four major constituents: αs1

, αs2, β, and κ. Each casein constituent affects how

kinesin adheres to the glass (or possibly how kinesin adheres to casein) and they ultimately affect the speed at which microtubules are observed to glide. Building on the work of Verma et. al.1, we have found that each constituent of bovine casein produces different outcomes in gliding assays.

ExperimentAll chemicals were purchased from Sigma unless stated otherwise. In order to determine the differences in how kinesin and microtubules interact dependent on casein constituents used as passivators, we first introduced caseins to our base buffer PEM [80mM PIPES (Sigma-80635), 1mM EGTA (Sigma-03778), 1mM MgCl2 (Sigma-M1028), pH 6.89] at0.5mg/mL. α casein, (a mixture of αs1

:αs2 ~ 7:22, Sigma-C6780), β casein

(Sigma-C6905), and κ casein (Sigma-C0406) go into solution easily with constant stirring, however, whole casein is notoriously difficult to get into solution. There exists a great deal of legacy procedures in the community on how to get whole casein into solution, some of which include extended periods of centrifugation and filtration. Whole casein does not have a secondary structure and will not denature under heat3. Thus, to get whole casein into PEM, we heat the solution and stir in 0.5mg/mL whole casein until it is completely dissolved since mild heating cannot affect the casein micelle structure. Finally, all casein solutions are passed through a 0.2µm filter to remove any possible bacteria that may be in the solution.

Results

20 µm

Beta casein

20 µm

Alpha casein

20 µm

Kappa casein

20 µm

Whole casein

1000mM Betaine

20 µm

2000mM Betaine

20 µm

"100%" D2Oκ casein passivation

20 µm

Squiggle

2000mM Betainα casein passivation

20 µm

Squiggles

700

650

600

550

500

450

Spee

d (n

m/s

)

100806040200

Concentration (%)

H218O

D2OFilled marker is the - end speedUnfilled marker is the + end speed

700

600

500

400

300

200

Spee

d (n

m/s

)

300025002000150010005000

Time (s)

Whole caseinAlpha caseinBeta caseinKappa casein

Filled marker is the - end speedUnfilled marker is the + end speed

Kinesin and microtubule in regular water.

Kinesin and microtubule in water with osmolytes.

Documents

Speed effects on gliding motility assays due to surface