Biomedical Engineering Reference
In-Depth Information
ilament.rotates.once..This.can.be.problematic.in.transport.since.the.cargo.will.be.rotating.
around.the.ilament,.increasing.the.likelihood.of.getting.stuck.on.a.neighboring.surface,.
being.knocked.off,.or.pulling.the.ilament.free.from.the.channel..In.some.circumstances,.
rotation. might. be. desired,. such. as. cargo. attachment. to. the. end. of. a. ilament. allowing. a.
freely.rotating.geometry.with.the.ilament.spinning. the.cargo.. Microtubules. in.contrast.
to.actin.do.not.encounter.this.problem.of.ilament.rotation.when.formed.with.the.typical.
13-protoilament.arrangement..In.this.arrangement,.protoilaments.run.straight.and.have.
no.helical.pitch.. Variations. from. the.13-protoilament.assembly.occur.in.nature.and.can.
also.arise.when.microtubules.are.nucleated.in.vitro.without.an.appropriate.template..In.
lieu.of.screening.microtubules.for.the.correct.13-fold.symmetry,.an.alternative.approach.
will. be. to. provide. nature's. template,. that. is,. the. gamma-tubulin. ring. complex. (γ-TuRC).
MTOC.complex.(Kollman.et.al.,.2010)..If.microtubule.dynamics.need.to.be.limited,.there.
are.many.possibilities.that.include,.microtubule.stabilizers.such.as.Taxol,.gentle.ixation.
by. cross-linking. with. glutaraldehyde. that. can. retain. kinesin. transport. abilities,. tubulin.
modiications,.genetic.engineering.of.the.tubulin.subunits.to.provide.microtubules.with.
altered. dynamic. parameters. that. include. reduced. catastrophe. (shift. from. growing. to.
shrinking),.or.novel.approaches.such.as.mimicking.ring.complexes.on.microtubules.
4.4.6 Cargo Attachment and release
The.complexity.of.how.to.sort.and.transport.various.cargos.begins.often.with.the.decision.
of.whether.to.functionalize.the.tail.or.stalk.of.a.motor.versus.manipulating.the.microtubule.
to.carry.out.the.transport.(Figure.4.4)..In.the.latter,.the.microtubule.will.be.driven.on.a.bed.
of.ixed.kinesin.motors.that.use.the.energy.of.ATP.hydrolysis.to.transport.the.microtubule..
The. consideration. here. is. how. to. attach. desired. cargos. to. the. microtubule. (Malcos. and.
Hancock,.2011)..It.is.important.to.design.in.features.of.high.afinity.and.high.speciicity..
The.reversible.bonds.for.attachment.will.have.a.speciic.lifetime.and.need.to.withstand.
forces. due. to. Brownian. motion,. which. require. bond. strengths. between. 1. pN. and. 1.nN..
An.example.of.how.Brownian.forces.affect.bonds.was.revealed.by.characterization.of.the.
noncovalent.bond.between.biotin.and.streptavidin..This.bond.can.withstand.a.force.of.5.pN.
for.1.min,.though.it.survives.for.only.100.ms.when.a.170.pN.force.is.applied.(Merkel.et.al.,.
1999)..The.size.of.cargo.that.can.be.applied.in.such.devices.varies,.from.nanometer.to.the.
microscale..In.one.example,.Hiyama.et.al..(2010).demonstrated.transport.using.liposomes.
as. cargo. that. varied. in. size. from. 100. to. 590.nm.. Though. fewer. numbers. of. larger. cargo.
were.transported,.the.larger.liposomes.carried.a.greater.total.volume.of.cargo.than.that.
of.the.smaller.liposomes.combined..Kinesin.easily.transports.supramolecular.structures.
with.a.diameter.of.20-50.nm,.such.as.synaptic.vesicles.(Cai.and.Sheng,.2009)..In.principle.
a.collective.force.generated.by.a.leet.of.kinesins.is.preferred.(Erickson.et.al..2011).and.can.
work.to.transport.small.microchips.up.to.20.μm.in.length.(Limberis.and.Stewart,.2000).
Functionalization. of. the. shuttle. component. is. key. to. being. able. to. attach. (and. detach).
any. form. of. cargo,. but. care. must. be. taken. not. to. disrupt. motility. by. compromising.
kinesin-microtubule.interactions..Biotinylation.is.a.common.method.used.to.functionalize.
the.surface.of.a.microtubule.to.enable.selective.binding.and.loading.of.cargo.(Hess.et.al.,.
2001).. This. is. done. by. covalently. bonding. biotin,. which,. in. turn,. allows. the. binding. of.
noncovalently.linked.molecules.such.as.streptavidin,.antibodies,.or.single-stranded.DNA.
(ssDNA).. The. predominant. strategy. used. to. attach. cargo. is. through. the. use. of. biotin/
avidin. (or. streptavidin). bridges,. antibody/antigen. interactions,. DNA. hybridization,.
or. polyhistidine. tags. (Figure. 4.5).. Two. methods. that. allow. incorporation. of. inorganics.
are. through. polyhistidine. tags,. which. bind. to. nickel. or. by. coating. quantum. dots. with.
