Isolation of newt lung ciliated cell models and characterization of their motile capabilities Public Deposited

http://ir.library.oregonstate.edu/concern/graduate_thesis_or_dissertations/tx31qn43g

Descriptions

Attribute NameValues
Creator
Abstract or Summary
  • The lack of a suitable demembranated cell model has hindered the study of the mechanisms responsible for the regulation of ciliary waveform and coordination. In this study procedures for the isolation of ciliated cells from the newt, Taricha granulosa, by trypsin dissociation, and their subsequent demembranation with Triton X-100 and reactivation with MgATP are described. Reactivation of these cell models with a high degree of mechanochemicalcoupling depends on avoidance of mechanical damage, and maintenance of optimal conditions during tyrypsinization, demembranation, and reactivation. Highly motile models were prepared from cells incubated in 0.5% trypsin for 48 hours at 4°C, treated briefly with 2 mM EDTA, separated by gentle agitation, and concentrated by centrifugation at low G forces. Optimal demembranation and reactivation conditions are similar to those described previously for isolated newt lung axonemes. Under these conditions, nearly 100% of the models can be reactivated when provided with MgATP, and 90-95% show coordinated beating in the ciliary tuft. Beat frequencies within the range measured in living cells can be obtained. The reactivated motility is stable for 30 minutes at constant MgATP. Ciliary waveform and the pattern of coordination are not different from those observed in living cells. On the basis of these results, it can be concluded that these cell models possess a high degree of mechanochemical coupling, and display motility that is characteristic of the living system. These highly coupled models were used to show that: 1) development of coordination in the ciliary tuft occurs at a higher substrate concentration range (10-25 μM) than that required to initiate motility per se (2-10 μM); 2) the outer dynein arms are essentially nonfunctional at substrate levels below 35 μM ATP, and they are not required for the coordinated beating of cilia; 3) ciliary waveforms and the type of metachrony are regulated independent of beat frequency; 4) beat frequency varies biphasically in response to MgATP, as has been shown previously for isolated, individual newt lung axonemes. Apparent Fmax and Km values of 25 Hz and 0.15 mM, and 35 Hz and 0.47 mM, respectively, have been obtained for each linear segment of the biphasic double reciprocal plot.
Resource Type
Date Available
Date Copyright
Date Issued
Degree Level
Degree Name
Degree Field
Degree Grantor
Commencement Year
Advisor
Committee Member
Academic Affiliation
Non-Academic Affiliation
Subject
Rights Statement
Peer Reviewed
Language
Digitization Specifications
  • File scanned at 300 ppi (Monochrome, 8-bit Grayscale) using ScandAll PRO 1.8.1 on a Fi-6670 in PDF format. CVista PdfCompressor 4.0 was used for pdf compression and textual OCR.
Replaces
Additional Information
  • description.provenance : Approved for entry into archive by Patricia Black(patricia.black@oregonstate.edu) on 2013-07-31T17:52:36Z (GMT) No. of bitstreams: 1 WeaverAlice1984.pdf: 1419909 bytes, checksum: 16d0a5c8ac8c6c6c4339d0a834f2822e (MD5)
  • description.provenance : Submitted by Katy Davis (kdscannerosu@gmail.com) on 2013-07-31T15:41:57Z No. of bitstreams: 1 WeaverAlice1984.pdf: 1419909 bytes, checksum: 16d0a5c8ac8c6c6c4339d0a834f2822e (MD5)
  • description.provenance : Approved for entry into archive by Patricia Black(patricia.black@oregonstate.edu) on 2013-08-05T16:37:07Z (GMT) No. of bitstreams: 1 WeaverAlice1984.pdf: 1419909 bytes, checksum: 16d0a5c8ac8c6c6c4339d0a834f2822e (MD5)
  • description.provenance : Made available in DSpace on 2013-08-05T16:37:07Z (GMT). No. of bitstreams: 1 WeaverAlice1984.pdf: 1419909 bytes, checksum: 16d0a5c8ac8c6c6c4339d0a834f2822e (MD5) Previous issue date: 1983-06-07

Relationships

Parents:

This work has no parents.

Last modified

Downloadable Content

Download PDF

Items