Photodamage and the Importance of Photoprotection in Biomolecular-Powered Device Applications

In recent years, an enhanced understanding of the mechanisms underlying photobleaching and photoblinking of fluorescent dyes has led to improved photoprotection strategies, such as reducing and oxidizing systems (ROXS) that reduce blinking and oxygen scavenging systems to reduce bleaching. Excitation of fluorescent dyes can also result in damage to catalytic proteins (e.g., biomolecular motors), affecting the performance of integrated devices. Here, we characterized the motility of microtubules driven by kinesin motor proteins using various photoprotection strategies, including a microfluidic deoxygenation device. Impaired motility of microtubules was observed at high excitation intensities in the absence of photoprotection as well as in the presence of an enzymatic oxygen scavenging system. In contrast, using a polydimethylsiloxane (PDMS) microfluidic deoxygenation device and ROXS, not only were the fluorophores slower to bleach but also moving the velocity and fraction of microtubules over time remained unaffected even at high excitation intensities. Further, we demonstrate the importance of photoprotection by examining the effect of photodamage on the behavior of a switchable mutant of kinesin. Overall, these results demonstrate that improved photoprotection strategies may have a profound impact on functional fluorescently labeled biomolecules in integrated devices

Mechanisms Underlying the Active Self-Assembly of Microtubule Rings and Spools

Active self-assembly offers a powerful route for the creation of dynamic multiscale structures that are presently inaccessible with standard microfabrication techniques. One such system uses the translation of microtubule filaments by surface-tethered kinesin to actively assemble nanocomposites with bundle, ring, and spool morphologies. Attempts to observe mechanisms involved in this active assembly system have been hampered by experimental difficulties with performing observation during buffer exchange and photodamage from fluorescent excitation. In the present work, we used a custom microfluidic device to remove these limitations and directly study ring/spool formation, including the earliest events (nucleation) that drive subsequent nanocomposite assembly. Three distinct formation events were observed: pinning, collisions, and induced curvature. Of these three, collisions accounted for the majority of event leading to ring/spool formation, while the rate of pinning was shown to be dependent on the amount of photodamage in the system. We further showed that formation mechanism directly affects the diameter and rotation direction of the resultant rings and spools. Overall, the fundamental understanding described in this work provides a foundation by which the properties of motor-driven, actively assembled nanocomposites may be tailored toward specific applications

Inhibition of Microtubule Depolymerization by Osmolytes

Microtubule dynamics play a critical role in the normal physiology of eukaryotic cells as well as a number of cancers and neurodegenerative disorders. The polymerization/depolymerization of microtubules is regulated by a variety of stabilizing and destabilizing factors, including microtubule-associated proteins and therapeutic agents (e.g., paclitaxel, nocodazole). Here we describe the ability of the osmolytes polyethylene glycol (PEG) and trimethylamine-<i>N</i>-oxide (TMAO) to inhibit the depolymerization of individual microtubule filaments for extended periods of time (up to 30 days). We further show that PEG stabilizes microtubules against both temperature- and calcium-induced depolymerization. Our results collectively suggest that the observed inhibition may be related to combination of the kosmotropic behavior and excluded volume/osmotic pressure effects associated with PEG and TMAO. Taken together with prior studies, our data suggest that the physiochemical properties of the local environment can regulate microtubule depolymerization and may potentially play an important role in in vivo microtubule dynamics

The Role of Membrane Fluidization in the Gel-Assisted Formation of Giant Polymersomes

<div><p>Polymersomes are being widely explored as synthetic analogs of lipid vesicles based on their enhanced stability and potential uses in a wide variety of applications in (e.g., drug delivery, cell analogs, etc.). Controlled formation of giant polymersomes for use in membrane studies and cell mimetic systems, however, is currently limited by low-yield production methodologies. Here, we describe for the first time, how the size distribution of giant poly(ethylene glycol)-poly(butadiene) (PEO-PBD) polymersomes formed by gel-assisted rehydration may be controlled based on membrane fluidization. We first show that the average diameter and size distribution of PEO-PBD polymersomes may be readily increased by increasing the temperature of the rehydration solution. Further, we describe a correlative relationship between polymersome size and membrane fluidization through the addition of sucrose during rehydration, enabling the formation of PEO-PBD polymersomes with a range of diameters, including giant-sized vesicles (>100 μm). This correlative relationship suggests that sucrose may function as a small molecule fluidizer during rehydration, enhancing polymer diffusivity during formation and increasing polymersome size. Overall the ability to easily regulate the size of PEO-PBD polymersomes based on membrane fluidity, either through temperature or fluidizers, has broadly applicability in areas including targeted therapeutic delivery and synthetic biology.</p></div

Effects of membrane fluidization on the formation of polymersomes.

<p>(a) Epifluorescence photomicrographs of PEO-PBD polymersomes formed on either agarose gels in water (left two images) or agarose gels in sucrose (right two images) and rehydrated in either water or sucrose as indicated at the top of the image. Scale bar = 10 μm. (b) Correlation of polymersome size (measured for over 80 polymersomes) and diffusion coefficients (calculated for at least five different polymersomes). X-error bars are standard error of the mean for the polymersome diameters. Y-error bars are standard error of the mean for the diffusion coefficients.</p

Dependency of vesicle size on different rehydration temperatures.

<p>PEO-PBD polymersomes were generated in water on 1% agarose gels for 30 min at varying temperatures on a hot plate. (a) Epifluorescence photomicrographs (scale bar = 10 μm), (b) average diameters (± standard error of the mean), and (c) frequency distribution plots for polymersomes formed at different temperatures.</p

Summary of the different polymers tested, their abbreviations used in the text and the molecular weight (M_w).

<p>Summary of the different polymers tested, their abbreviations used in the text and the molecular weight (M_w).</p

Diffusion coefficients (mean ± standard error) estimated from FRAP data for polymersomes formed from different polymers.

<p>Diffusion coefficients (mean ± standard error) estimated from FRAP data for polymersomes formed from different polymers.</p

Comparison of polymersomes formed by electroformation and gel-assisted rehydration.

<p>(a) Representative epifluorescence photomicrographs depicting PEO-PBD, positively-charged PEO-PBD (NH³⁺) and negatively-charged PEO-PBD (COO^-) polymersomes formation using platinum wire electroformation. (b) Epifluorescence photomicrographs depicting polymersome formation using gel-assisted rehydration after 1 h on an agarose gel at 40°C. Scale bar = 10 μm.</p

Fluorescence recovery after photobleaching (FRAP) analysis shows that polymersome membranes are fluid.

<p>(a) Epifluorescence imaging of a representative PEO-PBD polymersome pre-, during and post-fluorescence bleaching. The region of the membrane that was bleached is circled. Scale bar = 10 μm. (b) Time-dependent fluorescence recovery profiles for different polymers.</p