"Artificial mitotic spindle" generated by dielectrophoresis and protein micropatterning supports bidirectional transport of kinesin-coated beads

Maruti Uppalapati, Ying Ming Huang, Vidhya Aravamuthan, Thomas Nelson Jackson, William O. Hancock

Research output: Contribution to journalArticle

10 Citations (Scopus)

Abstract

The mitotic spindle is a dynamic assembly of microtubules and microtubule-associated proteins that controls the directed movement of chromosomes during cell division. Because proper segregation of the duplicated genome requires that each daughter cell receives precisely one copy of each chromosome, numerous overlapping mechanisms have evolved to ensure that every chromosome is transported to the cell equator during metaphase. However, due to the inherent redundancy in this system, cellular studies using gene knockdowns or small molecule inhibitors have an inherent limit in defining the sufficiency of precise molecular mechanisms as well as quantifying aspects of their mechanical performance. Thus, there exists a need for novel experimental approaches that reconstitute important aspects of the mitotic spindle in vitro. Here, we show that by microfabricating Cr electrodes on quartz substrates and micropatterning proteins on the electrode surfaces, AC electric fields can be used to assemble opposed bundles of aligned and uniformly oriented microtubules as found in the mitotic spindle. By immobilizing microtubule ends on each electrode, analogous to anchoring at centrosomes, solutions of motor or microtubule binding proteins can be introduced and their resulting dynamics analyzed. Using this "artificial mitotic spindle" we show that beads functionalized with plus-end kinesin motors move in an oscillatory manner analogous to the movements of chromosomes and severed chromosome arms during metaphase. Hence, features of directional instability, an established characteristic of metaphase chromosome dynamics, can be reconstituted in vitro using a pair of uniformly oriented microtubule bundles and a plus-end kinesin functionalized bead.

Original languageEnglish (US)
Pages (from-to)57-64
Number of pages8
JournalIntegrative Biology
Volume3
Issue number1
DOIs
StatePublished - Jan 1 2011

Fingerprint

Kinesin
Spindle Apparatus
Chromosomes
Electrophoresis
Microtubules
Metaphase
Electrodes
Proteins
Genes
Gene Knockdown Techniques
Microtubule Proteins
Centrosome
Quartz
Microtubule-Associated Proteins
Cell Division
Redundancy
Carrier Proteins
Cells
Electric fields
Genome

All Science Journal Classification (ASJC) codes

  • Biophysics
  • Biochemistry

Cite this

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title = "{"}Artificial mitotic spindle{"} generated by dielectrophoresis and protein micropatterning supports bidirectional transport of kinesin-coated beads",
abstract = "The mitotic spindle is a dynamic assembly of microtubules and microtubule-associated proteins that controls the directed movement of chromosomes during cell division. Because proper segregation of the duplicated genome requires that each daughter cell receives precisely one copy of each chromosome, numerous overlapping mechanisms have evolved to ensure that every chromosome is transported to the cell equator during metaphase. However, due to the inherent redundancy in this system, cellular studies using gene knockdowns or small molecule inhibitors have an inherent limit in defining the sufficiency of precise molecular mechanisms as well as quantifying aspects of their mechanical performance. Thus, there exists a need for novel experimental approaches that reconstitute important aspects of the mitotic spindle in vitro. Here, we show that by microfabricating Cr electrodes on quartz substrates and micropatterning proteins on the electrode surfaces, AC electric fields can be used to assemble opposed bundles of aligned and uniformly oriented microtubules as found in the mitotic spindle. By immobilizing microtubule ends on each electrode, analogous to anchoring at centrosomes, solutions of motor or microtubule binding proteins can be introduced and their resulting dynamics analyzed. Using this {"}artificial mitotic spindle{"} we show that beads functionalized with plus-end kinesin motors move in an oscillatory manner analogous to the movements of chromosomes and severed chromosome arms during metaphase. Hence, features of directional instability, an established characteristic of metaphase chromosome dynamics, can be reconstituted in vitro using a pair of uniformly oriented microtubule bundles and a plus-end kinesin functionalized bead.",
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"Artificial mitotic spindle" generated by dielectrophoresis and protein micropatterning supports bidirectional transport of kinesin-coated beads. / Uppalapati, Maruti; Huang, Ying Ming; Aravamuthan, Vidhya; Jackson, Thomas Nelson; Hancock, William O.

In: Integrative Biology, Vol. 3, No. 1, 01.01.2011, p. 57-64.

Research output: Contribution to journalArticle

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