Micro- and Nanostructures for Studies of Model Biological Systems

Abstract

Sub-cellular biological components are complex systems that work together to control and maintain cellular activity and health. It is useful to study the basic elements that constitute the sub-cellular components using model systems, which mimic certain

parts of the overall system, in order to better understand the system as a whole. Here we present a number of tools, which enable the study of a few model systems that mimic specific components of molecular motors and cellular membranes. The tools we use are nano- and microstructures that allow for the control and monitoring of two model biological systems: synthetic molecular motors and supported lipid bilayers, in order to probe their basic functions and interactions with the artificial environment.



The two synthetic motors of interest, that mimic cellular bipedal molecular motors, utilize either rectified diffusive motion or changes in motor conformation to take steps. The synthetic motors have been designed to isolate these aspects previously observed in natural bipedal motor stepping. We have fabricated and tested microand nanofluidic devices which make it possible to control the motors’ stepping with simultaneous observation using optical microcopy. Additionally, these devices are designed to enable testing of the motors’ performance against a load force. We have

also used a microfluidics device with a natural helicase motor that moves along DNA, in much the same way as one of the synthetic motors is designed to do. In this study we have successfully controlled the motor motion with addition and withdrawal of the motor’s fuel supply, ATP, in the surrounding solution, and we have monitored single protein motors as they move and stop along the DNA track. These devices and techniques set the stage for future measurements of the synthetic molecular motors. Additionally, these fluidic devices have a number of other potential applications in single-molecule studies, as they allow for the changing of the chemical environment surrounding single-molecules whilst causing little disturbance to these. Supported lipid bilayers are a simplified model version of the complex cell membrane, which contains not only lipids, but also a number of other intra-membrane components needed for cellular function. We have used a substrate with a large number of vertical nanowires standing parallel to one another to study the bilayer interaction on a highly curved support. This type of support has interesting

applications in the field of localized injection into cells. In this application it is still unclear how the cellular membrane responds to the nanowire supports, and how injection can occur. Using the nanowire devices, we have formed a supported lipid bilayer that follows the nanowires, which could allow for future studies focusing on the interaction between lipid membranes and the nanowires. Additionally, because the membrane follows the nanowires, this device could be used as a platform to study

lipids at high curvature, for example membrane components, such as proteins, that favor high curvature.



Each of the devices we have developed will hopefully bring insight into the working of these model biological systems, which in turn could reveal information about how components of the natural sub-cellular system are functioning.

Abstract (Swedish)

Popular Abstract in English

Small Devices to Study Small Biology

Biological components that are smaller than a cell are difficult to study directly. A great deal of work has been done developing devices and techniques that allow scientists to control and observe sub-cellular biology. In this thesis I, along with my colleagues, have made a number of devices that give us control over the small biological components that we are studying. All of these devices are designed to study simplified versions of natural biological systems, namely motor proteins and cell membranes. The simplified versions allow us to isolate certain components of the more complex natural system and the devices give us a method for controlling the components on the nano- and micro-size-scale. By studying the isolated components, we are learning about the natural systems. In the future, this information may help scientists understand the natural systems that are central to cellular function, or the information learned may contribute to bioengineering in creating new systems.



Controlling Artificial Biological Motors

Our devices, can fit in the palm of your hand, like the one shown in the picture to the right, which is filled with blue food coloring. They have plumbing with pipes the size of your hair, ~100 µm, and pipes 1000X smaller. The large tubes connected to the small pipes make it easier to push fluid into the small pipes.



We designed the pipes so that we can control the water flow surrounding proteins and DNA. We need to be able to change from one fluid to the next fluid that surrounds the protein or DNA, so we pump in one fluid after another, like the drawing shown in Figure 1.



We can use these devices to control biological molecular motors found in nature, and simplified artificial versions of those motors. Molecular motors are small machines that can use the chemicals surrounding them as fuel and in turn perform mechanical work. Motors inside cells have many different jobs. Some act like pumps to move ions from one side of a membrane to another. Some work together to repair damaged DNA, or produce more proteins. Others can move cargo from one place to another; they have two coordinated feet that step along a track inside the cell.



Artificial motors have been designed as well. These are molecular motors that mimic nature, but they are made in the lab. Two motor designs that we are using have a body and feet, which move along a linear track. All of these components are made of DNA and proteins. One of the motors takes steps like an inchworm (illustrated in Figure 2) and the other, which has three feet, moves like a gymnast doing one-handed cartwheels; we call it the tumbleweed (illustrated in Figure 3). In both cases we can control when the motor takes steps by changing the fluid surrounding it.



The proteins can stick to the DNA in certain places when certain particles are in the fluid around it. By controlling which fluid mixture is surrounding the proteins and DNA in the pipes at certain times, we can make certain proteins stick to certain pieces of DNA. By coordinating which fluid is in the pipe at a given time, we can make the motor move in one direction along a track. In this thesis we have focused on the design of the pipes needed to change between different fluids. We have designed and made a variety of devices, which each have unique features for each motor design. We have measured the time it takes to change between fluids to be a fifth of a second.



Changing Fluids in Small Pipes

Water behaves differently in nano-sized and micro-sized pipes. Small molecules move in water as if it were syrup. We utilize this property of water on the small scale to control the fluid surrounding the protein and DNA in the pipes. The solute in the fluid moves between streams of fluid only due to random thermal movement, otherwise two streams of fluid next to each other in one pipe stay parallel and separate. In one device we have two pipes with different fluids that connect in a Y shape, creating two streams of fluid in one pipe as is shown in Figure 4 (top), and by increasing the pressure on the inlet to one pipe we created unequal parallel streams, like in Figure 4 (bottom).



We have used this device to look at a natural motor protein, a helicase motor that unwinds DNA. We dissolved the fuel for the motor in one fluid and left the other without fuel. Alternating between the top inlet and bottom inlet having the higher pressure, we changed which fluid is in the center of the channel. This is how we turned the fuel on and off for the natural helicase motor. We then watched, using a microscope, as the motor moved, unwinding the DNA, and stopped. The proteins, which are ~10 nm in size, are too small to see, so we attached bright particles to the proteins so that we could shine light of a particular color at the particles and the particles shone back with another color, through the microscope, at the camera.



We used similar principles of fluid behavior to design a more complicated device for switching between three fluids, like what is shown in Figure 1, and plan to use this with the tumbleweed motor in the future. In the device used to control the inchworm motor, a device like the one used for the tumbleweed motor was connected to nano-pipes. Due to the small connections between the micro-size and nano-size pipes we were able to change between fluids without having a fluid flow inside the nano-pipes, which would push backward on the motor.



Mimicking Cell Membranes on Nano-Pillars

Another device we have used has many pillars that are about 0.06 µm wide and around 3 µm long. Similar pillars have previously been used as nano-needles for injection into individual cells. However, it seems the needles do not pierce the cells, but rather the membrane around the cell surrounds the needles. We have used the nano-pillars with a simplified membrane to investigate this interaction. We find that the membrane, on its own, follows along the surface of the pillars like the drawing in Figure 5 A, rather than spanning on top of the pillars like Figure 5 B.

Figure 5 (A) A model cell membrane, in pink, following along the surface of the nano-pillars, in blue. This is the conformation we observe experimentally.

(B) Alternative membrane conformation, where the membrane sits on top of the nano-pillars. These are not observed experimentally.



To study the membrane on the nano-pillars, we used a microscope that can image in 3D, a confocal microscope. The membrane, labeled with bright particles, follows the vertical pillar geometry, like Figure 5 A. We also know that the membrane is made of many small molecules that move freely within the membrane sheet. To find the rate at which they move, we labeled some of the membrane molecules with bright molecules. We then damaged a small region so that the labeled molecules no longer shone, and we watched as the bright molecules moved to the dark regions and vice versa. By fitting the overall rate of change of the brightness in this region to a theoretical curve, we found the rate of movement of the particles. This rate should not change, whether the membrane is on a flat support or on one with pillars. After adjusting the theory to fit the scenarios drawn in Figure 5 A or B, we saw a systematic change in the rate for scenario B. There was almost no change in the rate for scenario A, which means scenario A is most likely. This result matched what others have seen with cell membranes.



Prospective

Each device presented here has been developed for a specific small-scale biological system, but each one also has other possible applications. The small pipes can be used with the natural and artificial motors as described, but beyond that there are a plethora of biological systems where one would like to control the fluid surrounding the proteins, DNA, or cells. As we dive deeper into more complex biological systems, more control over the fluid is desirable. We imagine using the membrane/nano-pillar device to study proteins or membrane components that prefer to be at regions of high curvature. The device could also be a platform for investigating membrane permeability at high curvature, which has been proposed to be the mechanism for observed cell injection with nano-needles. All of these devices have potential for further use in studying small-scale natural and artificial biological components beyond what is presented here and possibly even for applications in future diagnostic devices for medical applications.



<<Figure 1 A side view of a micro-pipe. The fluid flow is from left to right and the red, blue and teal fluids pass over a protein on the bottom of the pipe in a particular order.>>



<<Figure 2 An artificial motor with a DNA body (black) and feet (green and pink). By changing the particles in the surrounding fluid, the feet stick to the surrounding proteins (stuck to the surface) and the length of the DNA changes. By coordinating the fluid changes, the motor moves to the right like an inchworm.>>



<<Figure 3 An artificial protein motor with three feet, pink, blue and green that stick to a DNA track when certain particles are in the surrounding fluid. When the particles enter in the correct order, the protein steps to the right, turning like a tumbleweed. >>



<<Figure 4 (top) A Y shaped micro-pipe with two different fluids coming in from the left with inlet pressure P and outlet pressure P0 resulting in two parallel and equal sized fluid streams. (bottom) The pressure on the top inlet is increased to P+ so the corresponding flow stream is wider in the outlet pipe.>>

• author: Niman, Cassandra
• supervisor: Heiner Linke
• opponent: Turberfield, Andrew, University of Oxford, United Kingdom
• organization: Solid State Physics; NanoLund: Centre for Nanoscience
• publishing date: 2014
• type: Thesis
• publication status: published
• subject: Condensed Matter Physics
• keywords: Fysicumarkivet A:2014:Niman
• defense location: Lecture hall Rydbergsalen, Department of Physics, Sölvegatan 14, Lund University, Faculty of Engineering LTH
• defense date: 2014-11-21 09:15:00
• ISBN: 978-91-7623-115-9
• language: English
• LU publication?: yes
• id: 03d48a10-0992-4cb5-83eb-1d150d7e3961 (old id 4731266)
• date added to LUP: 2016-04-04 14:11:03
• date last changed: 2020-09-16 11:16:24