17 research outputs found
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
Single Filament Behavior of Microtubules in the Presence of Added Divalent Counterions
Microtubules (MTs) are hollow biopolymeric
filaments that function
to define the shape of eukaryotic cells, serve as a platform for intracellular
vesicular transport, and separate chromosomes during mitosis. One
means of physiological regulation of MT mechanics and dynamics, critical
to their adaptability in such processes, is through electrostatics
due to the strong polyelectrolyte nature of MTs. Here, we show that
in the presence of physiologically relevant amounts of divalent salts,
MTs experience a dramatic increase in persistence length or stiffness,
which is counter to theoretical expectations and experimental observations
in similar systems (e.g., DNA). Divalent salt-dependent effects on
MT dynamics were also observed with respect to suppressing depolymerization
as well as reducing dispersion in kinesin-driven molecular motor transport
assays. These results establish a novel mechanism by which MT dynamics,
mechanics, and interaction with molecular motors may be regulated
by physiologically relevant concentrations of divalent salts
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
A Continuous Network of Lipid Nanotubes Fabricated from the Gliding Motility of Kinesin Powered Microtubule Filaments
Synthetic interconnected lipid nanotube
networks were fabricated
on the millimeter scale based on the simple, cooperative interaction
between phospholipid vesicles and kinesin–microtubule (MT)
transport systems. More specifically, taxol-stabilized MTs, in constant
2D motion via surface absorbed kinesin, extracted and extended lipid
nanotube networks from large L<sub>α</sub> phase multilamellar
liposomes (5–25 μm). Based on the properties of the inverted
motility geometry, the total size of these nanofluidic networks was
limited by MT surface density, molecular motor energy source (ATP),
and total amount and physical properties of lipid source material.
Interactions between MTs and extended lipid nanotubes resulted in
bifurcation of the nanotubes and ultimately the generation of highly
branched networks of fluidically connected nanotubes. The network
bifurcation was easily tuned by changing the density of microtubules
on the surface to increase or decrease the frequency of branching.
The ability of these networks to capture nanomaterials at the membrane
surface with high fidelity was subsequently demonstrated using quantum
dots as a model system. The diffusive transport of quantum dots was
also characterized with respect to using these nanotube networks for
mass transport applications
A Continuous Network of Lipid Nanotubes Fabricated from the Gliding Motility of Kinesin Powered Microtubule Filaments
Synthetic interconnected lipid nanotube
networks were fabricated
on the millimeter scale based on the simple, cooperative interaction
between phospholipid vesicles and kinesin–microtubule (MT)
transport systems. More specifically, taxol-stabilized MTs, in constant
2D motion via surface absorbed kinesin, extracted and extended lipid
nanotube networks from large L<sub>α</sub> phase multilamellar
liposomes (5–25 μm). Based on the properties of the inverted
motility geometry, the total size of these nanofluidic networks was
limited by MT surface density, molecular motor energy source (ATP),
and total amount and physical properties of lipid source material.
Interactions between MTs and extended lipid nanotubes resulted in
bifurcation of the nanotubes and ultimately the generation of highly
branched networks of fluidically connected nanotubes. The network
bifurcation was easily tuned by changing the density of microtubules
on the surface to increase or decrease the frequency of branching.
The ability of these networks to capture nanomaterials at the membrane
surface with high fidelity was subsequently demonstrated using quantum
dots as a model system. The diffusive transport of quantum dots was
also characterized with respect to using these nanotube networks for
mass transport applications
Templated Nanocrystal Assembly on Biodynamic Artificial Microtubule Asters
Microtubules (MTs) and the MT-associated proteins (MAPs) are critical cooperative agents involved in complex nanoassembly processes in biological systems. These biological materials and processes serve as important inspiration in developing new strategies for the assembly of synthetic nanomaterials in emerging techologies. Here, we explore a dynamic biofabrication process, modeled after the form and function of natural aster-like MT assemblies such as centrosomes. Specifically, we exploit the cooperative assembly of MTs and MAPs to form artificial microtubule asters and demonstrate that (1) these three-dimensional biomimetic microtubule asters can be controllably, reversibly assembled and (2) they serve as unique, dynamic biotemplates for the organization of secondary nanomaterials. We describe the MAP-mediated assembly and growth of functionalized MTs onto synthetic particles, the dynamic character of the assembled asters, and the application of these structures as templates for three-dimensional nanocrystal organization across multiple length scales. This biomediated nanomaterials assembly strategy illuminates a promising new pathway toward next-generation nanocomposite development
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
Engineering Lipid Structure for Recognition of the Liquid Ordered Membrane Phase
The
selective partitioning of lipid components in phase-separated
membranes is essential for domain formation involved in cellular processes.
Identifying and tracking the movement of lipids in cellular systems
would be improved if we understood how to achieve selective affinity
between fluorophore-labeled lipids and membrane assemblies. Here,
we investigated the structure and chemistry of membrane lipids to
evaluate lipid designs that partition to the liquid ordered (L<sub>o</sub>) phase. A range of fluorophores at the headgroup position
and lengths of PEG spacer between the lipid backbone and fluorophore
were examined. On a lipid body with saturated palmityl or palmitoyl
tails, we found that although the lipid tails can direct selective
partitioning to the L<sub>o</sub> phase through favorable packing
interactions, headgroup hydrophobicity can override the partitioning
behavior and direct the lipid to the disordered membrane phase (L<sub>d</sub>). The PEG spacer can serve as a buffer to mute headgroup–membrane
interactions and thus improve L<sub>o</sub> phase partitioning, but
its effect is limited with strongly hydrophobic fluorophore headgroups.
We present a series of lipid designs leading to the development of
novel fluorescently labeled lipids with selective affinity for the
L<sub>o</sub> phase
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 ÎĽm). 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
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