CHAPTER
Microtubules, MAPs, and motor patterns
2
Kasimira T. Stanhope, Jennifer L. Ross1 Molecular and Cellular Biology Graduate Program, Department of Physics, University of Massachusetts Amherst, Amherst, MA, USA 1
Corresponding author: E-mail: [email protected]
CHAPTER OUTLINE Introduction .............................................................................................................. 24 1. Methods .............................................................................................................. 25 2. Simple Filament-gliding Assay with Kinesin-1 and Microtubules ............................. 25 2.1 Flow Chambers ..................................................................................... 25 2.1.1 Materials ............................................................................................ 25 2.1.2 Chamber construction ........................................................................ 25 2.1.3 Notes ................................................................................................. 25 2.2 Gliding Assay Reagents and Buffers........................................................ 27 2.3 Experimental Details ............................................................................. 28 2.4 Analysis and Notes ................................................................................ 28 2.4.1 Kymographs ....................................................................................... 28 2.4.2 Filament end tracking ......................................................................... 31 2.4.3 Notes ................................................................................................. 31 3. Gliding Assay with MAP65 Cross-linkers to Visualize Dynamic Cross-linking ........... 31 3.1 MAP65 Purification ............................................................................... 31 3.1.1 Preparation reagents and buffers ........................................................ 31 3.1.2 Protein purification ............................................................................. 32 3.2 Gliding Assay Buffers ............................................................................ 34 3.3 Experimental Details ............................................................................. 34 3.4 Analysis and Notes ................................................................................ 34 4. Cell-like Patterns from Gliding Prebundled Microtubule Filaments........................... 35 4.1 Reagents and Buffers ............................................................................ 35 4.2 Prebundling Microtubules ...................................................................... 35 4.3 Experimental Details ............................................................................. 35 Discussion and Summary........................................................................................... 36 Acknowledgments ..................................................................................................... 37 References ............................................................................................................... 37 Methods in Cell Biology, Volume 128, ISSN 0091-679X, http://dx.doi.org/10.1016/bs.mcb.2015.02.003 © 2015 Elsevier Inc. All rights reserved.
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CHAPTER 2 Microtubules, MAPs, and motor patterns
Abstract Cells have an amazing ability to self-organize and rearrange their interiors. Such morphology changes are essential to cell development, division, and motility. The core of a cell’s internal organization lies with the cytoskeleton made of both microtubule and actin filaments with their associated proteins and ATP-utilizing enzymes. Despite years of in vitro reconstitution experiments, we still do not fully understand how the cytoskeleton can self-organize. In an attempt to create a simple system of selforganization, we have used a simple filament-gliding assay to examine how kinesin-1driven motion of microtubules can generate cell-like organization in the presence of excess filaments and antiparallel cross-linkers.
INTRODUCTION The cell is an inherently nonequilibrium environment where countless nanoscale machines, called enzymes, use energy to perform work opposing the entropic mixing force of diffusion. Such machines work in concert to push and pull biological molecules, macromolecules, and networks to where they need to be in time and space. No place is this energetic dance more obvious than in the cytoskeleton. Perhaps this is because the cytoskeleton organizes the cell’s interior, is used as the highway system to transport goods and services, and obviously and dramatically rearranges during cell division, motility, and development. Whatever the reason, groups have been studying cytoskeletal organization in cells for decades (Kirschner & Schulze, 1986; Ne´de´lec, Surrey, & Karsenti, 2003; Dogterom & Surrey, 2013). In vitro reconstitution experiments of cytoskeleton organization using purified components have been worked on for many years (Dogterom & Surrey, 2013). Several groups have worked on this problem, which is rich and deep due to the extensive number of cytoskeleton binding, cross-linking, and translocating proteins. Many of the published procedures involve difficult steps including nanofabrication (Ne´de´lec, Surrey, Maggs, & Leibler, 1997; Laan & Dogterom, 2010), use of extracts with many unknown factors (Cahu & Surrey, 2009; Pinot et al., 2009; Brugue´s, Nuzzo, Mazur, & Needleman, 2012), and high-end microscopy methods (Surrey, Nedelec, Leibler, & Karsenti, 2001; Brugue´s et al., 2012). The difficulty to performing these experiments has limited the number of researchers working on the problem and made reproducing prior results difficult. In an effort to simplify the system to something reproducible and easy to perform, we decided to modify a simple filament-gliding assay. In this chapter, we describe our simple filament-gliding assay and then add modifications to the assay including adding cross-linking proteins and more filaments. Finally, we describe a system that recapitulates cell-like microtubule organizations similar to those found in mitosis using only three protein components.
2. Simple filament-gliding assay with kinesin-1 and microtubules
1. METHODS Here, we outline the experimental methods to create cell-like patterns in vitro based on a simple microtubule-gliding assay powered by kinesin-1 motor proteins (Liu, Tu¨zel, & Ross, 2011; Pringle et al., 2013). We systematically add more microtubules or other types of associated proteins to probe how the patterns change in increased complexity. The microtubule-associated protein (MAP) we have used are the plant antiparallel cross-linker, MAP65-1. This is the plant homolog of PRC-1 (mammalian) or Ase1 (yeast).
2. SIMPLE FILAMENT-GLIDING ASSAY WITH KINESIN-1 AND MICROTUBULES The gliding assay with microtubules and kinesin-1 is a simple assay that undergraduates can do in any lab. No special chambers or glass treatment is needed. Here, we describe the flow chambers, reagents and buffers, and how the experiment is performed.
2.1 FLOW CHAMBERS 2.1.1 Materials Coverslip (22 22 1.5 mm, No. 1.5, Thermo Fisher Scientific). Coverslip (22 30 1.5 mm, No. 1.5, Thermo Fisher Scientific). Glass slide (25 75 1 mm, No. 1.5, Thermo Fisher Scientific). Permanent double-sided clear plastic tape (3 M). 5-min Zpoxy epoxy (Pacer).
2.1.2 Chamber construction 1. Clean cover glass and coverslip with double distilled water and ethanol. Dry with a kimwipe and leave under a petri dish to protect from dust. 2. Place double-sided tape 4e5 mm apart on the cover glass creating a horizontal flow chamber (Figure 1(A)). 3. Place 22 22 mm coverslip on top of the tape and press down to seal. Press only on the tape and not in the middle of the path because you can crack the cover glass. When pressing on the tape, it should become more transparent indicating that the chamber is sealed (Figure 1(A) and (C)).
2.1.3 Notes The chamber is 0.1 mm deep because the double stick tape is 100 mm thick. The cover glass is 22 mm long, setting the length of the flow path. These two lengths are fixed, so the volume of the chamber is totally determined by the width of the flow path between the two pieces of double stick tape. To make a 10 mL chamber volume, place the pieces of tape 4.5 mm apart.
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CHAPTER 2 Microtubules, MAPs, and motor patterns
FIGURE 1 Simple filament-gliding assay chamber construction and use. (A) Typical flow chamber with flow path parallel to long axis of slide. The top shows a schematic where the tape is gray and the flow path is denoted in blue (dark gray in print versions). The bottom shows the actual chamber with a sealed tape that is transparent. (B) Cross-flow chamber uses a longer cover glass with the flow direction perpendicular to the slide long axis enabling the flow path to be accessible when the cover glass is down on an inverted microscope. The top shows a schematic with the tape in gray and the flow path in blue (dark gray in print versions). The bottom shows the actual sealed chamber. (C) Example of a chamber where the double-sided tape making the flow path is not sealed. Comparing the look of the tape to that in (A), it is clear that the tape is less transparent and thus not making contact with both the cover glass and the slide. (D) Schematic of the side view of the flow chamber with the cover glass down on an inverted microscope. (E) Schematic of the surface treatment of the flow chamber in order to perform a gliding assay. Kinesin coats the cover glass first. The microtubules are flowed in later to bind to the kinesin. The kinesin motors walk to the plus-end pushing the microtubule with the minus-end forward. (F) Time series of a gliding assay. Two clear filaments gliding are denoted by arrowheads. The time between frames is 55 s. The scale bar on the last frame is 5 mm.
2. Simple filament-gliding assay with kinesin-1 and microtubules
The chambers are used coverslip side down on a modern inverted microscope for epifluorescence. This makes it difficult to flow in more reagents while imaging. To have a chamber with an accessible flow path, you can build a cross-flow chamber with a 22 30 mm coverslip perpendicular to the slide (Figure 1(B)). When the cross-flow chamber is placed on the inverted microscope, you can still pipette into the chamber. We often work with the chamber open at the ends of the flow path because we do not image very long (
Microtubules, MAPs, and motor patterns
2
Kasimira T. Stanhope, Jennifer L. Ross1 Molecular and Cellular Biology Graduate Program, Department of Physics, University of Massachusetts Amherst, Amherst, MA, USA 1
Corresponding author: E-mail: [email protected]
CHAPTER OUTLINE Introduction .............................................................................................................. 24 1. Methods .............................................................................................................. 25 2. Simple Filament-gliding Assay with Kinesin-1 and Microtubules ............................. 25 2.1 Flow Chambers ..................................................................................... 25 2.1.1 Materials ............................................................................................ 25 2.1.2 Chamber construction ........................................................................ 25 2.1.3 Notes ................................................................................................. 25 2.2 Gliding Assay Reagents and Buffers........................................................ 27 2.3 Experimental Details ............................................................................. 28 2.4 Analysis and Notes ................................................................................ 28 2.4.1 Kymographs ....................................................................................... 28 2.4.2 Filament end tracking ......................................................................... 31 2.4.3 Notes ................................................................................................. 31 3. Gliding Assay with MAP65 Cross-linkers to Visualize Dynamic Cross-linking ........... 31 3.1 MAP65 Purification ............................................................................... 31 3.1.1 Preparation reagents and buffers ........................................................ 31 3.1.2 Protein purification ............................................................................. 32 3.2 Gliding Assay Buffers ............................................................................ 34 3.3 Experimental Details ............................................................................. 34 3.4 Analysis and Notes ................................................................................ 34 4. Cell-like Patterns from Gliding Prebundled Microtubule Filaments........................... 35 4.1 Reagents and Buffers ............................................................................ 35 4.2 Prebundling Microtubules ...................................................................... 35 4.3 Experimental Details ............................................................................. 35 Discussion and Summary........................................................................................... 36 Acknowledgments ..................................................................................................... 37 References ............................................................................................................... 37 Methods in Cell Biology, Volume 128, ISSN 0091-679X, http://dx.doi.org/10.1016/bs.mcb.2015.02.003 © 2015 Elsevier Inc. All rights reserved.
23
24
CHAPTER 2 Microtubules, MAPs, and motor patterns
Abstract Cells have an amazing ability to self-organize and rearrange their interiors. Such morphology changes are essential to cell development, division, and motility. The core of a cell’s internal organization lies with the cytoskeleton made of both microtubule and actin filaments with their associated proteins and ATP-utilizing enzymes. Despite years of in vitro reconstitution experiments, we still do not fully understand how the cytoskeleton can self-organize. In an attempt to create a simple system of selforganization, we have used a simple filament-gliding assay to examine how kinesin-1driven motion of microtubules can generate cell-like organization in the presence of excess filaments and antiparallel cross-linkers.
INTRODUCTION The cell is an inherently nonequilibrium environment where countless nanoscale machines, called enzymes, use energy to perform work opposing the entropic mixing force of diffusion. Such machines work in concert to push and pull biological molecules, macromolecules, and networks to where they need to be in time and space. No place is this energetic dance more obvious than in the cytoskeleton. Perhaps this is because the cytoskeleton organizes the cell’s interior, is used as the highway system to transport goods and services, and obviously and dramatically rearranges during cell division, motility, and development. Whatever the reason, groups have been studying cytoskeletal organization in cells for decades (Kirschner & Schulze, 1986; Ne´de´lec, Surrey, & Karsenti, 2003; Dogterom & Surrey, 2013). In vitro reconstitution experiments of cytoskeleton organization using purified components have been worked on for many years (Dogterom & Surrey, 2013). Several groups have worked on this problem, which is rich and deep due to the extensive number of cytoskeleton binding, cross-linking, and translocating proteins. Many of the published procedures involve difficult steps including nanofabrication (Ne´de´lec, Surrey, Maggs, & Leibler, 1997; Laan & Dogterom, 2010), use of extracts with many unknown factors (Cahu & Surrey, 2009; Pinot et al., 2009; Brugue´s, Nuzzo, Mazur, & Needleman, 2012), and high-end microscopy methods (Surrey, Nedelec, Leibler, & Karsenti, 2001; Brugue´s et al., 2012). The difficulty to performing these experiments has limited the number of researchers working on the problem and made reproducing prior results difficult. In an effort to simplify the system to something reproducible and easy to perform, we decided to modify a simple filament-gliding assay. In this chapter, we describe our simple filament-gliding assay and then add modifications to the assay including adding cross-linking proteins and more filaments. Finally, we describe a system that recapitulates cell-like microtubule organizations similar to those found in mitosis using only three protein components.
2. Simple filament-gliding assay with kinesin-1 and microtubules
1. METHODS Here, we outline the experimental methods to create cell-like patterns in vitro based on a simple microtubule-gliding assay powered by kinesin-1 motor proteins (Liu, Tu¨zel, & Ross, 2011; Pringle et al., 2013). We systematically add more microtubules or other types of associated proteins to probe how the patterns change in increased complexity. The microtubule-associated protein (MAP) we have used are the plant antiparallel cross-linker, MAP65-1. This is the plant homolog of PRC-1 (mammalian) or Ase1 (yeast).
2. SIMPLE FILAMENT-GLIDING ASSAY WITH KINESIN-1 AND MICROTUBULES The gliding assay with microtubules and kinesin-1 is a simple assay that undergraduates can do in any lab. No special chambers or glass treatment is needed. Here, we describe the flow chambers, reagents and buffers, and how the experiment is performed.
2.1 FLOW CHAMBERS 2.1.1 Materials Coverslip (22 22 1.5 mm, No. 1.5, Thermo Fisher Scientific). Coverslip (22 30 1.5 mm, No. 1.5, Thermo Fisher Scientific). Glass slide (25 75 1 mm, No. 1.5, Thermo Fisher Scientific). Permanent double-sided clear plastic tape (3 M). 5-min Zpoxy epoxy (Pacer).
2.1.2 Chamber construction 1. Clean cover glass and coverslip with double distilled water and ethanol. Dry with a kimwipe and leave under a petri dish to protect from dust. 2. Place double-sided tape 4e5 mm apart on the cover glass creating a horizontal flow chamber (Figure 1(A)). 3. Place 22 22 mm coverslip on top of the tape and press down to seal. Press only on the tape and not in the middle of the path because you can crack the cover glass. When pressing on the tape, it should become more transparent indicating that the chamber is sealed (Figure 1(A) and (C)).
2.1.3 Notes The chamber is 0.1 mm deep because the double stick tape is 100 mm thick. The cover glass is 22 mm long, setting the length of the flow path. These two lengths are fixed, so the volume of the chamber is totally determined by the width of the flow path between the two pieces of double stick tape. To make a 10 mL chamber volume, place the pieces of tape 4.5 mm apart.
25
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CHAPTER 2 Microtubules, MAPs, and motor patterns
FIGURE 1 Simple filament-gliding assay chamber construction and use. (A) Typical flow chamber with flow path parallel to long axis of slide. The top shows a schematic where the tape is gray and the flow path is denoted in blue (dark gray in print versions). The bottom shows the actual chamber with a sealed tape that is transparent. (B) Cross-flow chamber uses a longer cover glass with the flow direction perpendicular to the slide long axis enabling the flow path to be accessible when the cover glass is down on an inverted microscope. The top shows a schematic with the tape in gray and the flow path in blue (dark gray in print versions). The bottom shows the actual sealed chamber. (C) Example of a chamber where the double-sided tape making the flow path is not sealed. Comparing the look of the tape to that in (A), it is clear that the tape is less transparent and thus not making contact with both the cover glass and the slide. (D) Schematic of the side view of the flow chamber with the cover glass down on an inverted microscope. (E) Schematic of the surface treatment of the flow chamber in order to perform a gliding assay. Kinesin coats the cover glass first. The microtubules are flowed in later to bind to the kinesin. The kinesin motors walk to the plus-end pushing the microtubule with the minus-end forward. (F) Time series of a gliding assay. Two clear filaments gliding are denoted by arrowheads. The time between frames is 55 s. The scale bar on the last frame is 5 mm.
2. Simple filament-gliding assay with kinesin-1 and microtubules
The chambers are used coverslip side down on a modern inverted microscope for epifluorescence. This makes it difficult to flow in more reagents while imaging. To have a chamber with an accessible flow path, you can build a cross-flow chamber with a 22 30 mm coverslip perpendicular to the slide (Figure 1(B)). When the cross-flow chamber is placed on the inverted microscope, you can still pipette into the chamber. We often work with the chamber open at the ends of the flow path because we do not image very long (
