Investigating Ciliary Beat Patterns through Correlated AFM, Fluorescence and Electron Microscopy: Structure, Dynamics, ForcesCISMM: Computer Integrated Systems for Microscopy and Manipulation

Collaborators: Larry Ostrowski (School of Medicine)

Project Lead: Michael Falvo Investigators: Atsuko Negishi, Robert Wontsetler (School of Medicine) ,

http://cs.unc.edu/Research/nano/cismm/cystic

Poster Date

Motivation and Objectives: Cross Platform Investigation of CiliaThe underlying mechanism of ciliary beat patterns remains a fascinating puzzle of profound practical importance. The mechanical operation of cilia is central to the understanding of diseases such as primary ciliary dyskinesia and cystic fibrosis. The mucociliary clearance system is the first line of defense against inhaled particulates, aerosols, and pathogens in the airways of the lung. Besides propelling the mucus layer in the lungs, cilia play other important roles in the body including bilateral orientation in embryos and propulsion of ovum in fallopian tubes.

This collaboration is driving the research to understand the underlying mechanism of ciliary beat patterns. While important insights have been gained from modeling and experiments in flagella, the mechanical operation of the cilium consists of control systems that are at present very poorly understood. The coordination of thousands of motors in highly organized structures perhaps represents the next scale of challenge for detailed understanding, from the molecular scale to the superstructure. We are characterizing cilia beating by correlating structural, dynamic and force data of cilia using a hybrid microscopy system.

Ciliary “Tassels”: Tapered distal end structure of cilia. Left panel is AFM data of isolated cilia. Upper panel shows SEM data of cilia still attached to a cultured epithelial cell. To the best of our knowledge, little is known about the structure or function of these cilia ends. Located at the distal end, they are involved in the mechanical coupling of the cilium to their hydrodynamic environment (mucus in the case of the lung epithelial cilia). We will investigate this distal end structure through correlated and simultaneous microscopy as outlined in the project description. A combination of our structural and dynamics studies in which the isolated cilia are reactivated may lend valuable insight into the mechanical function of these structures.

Correlated AFM/SEM of Cilia. The figure to the left shows correlated AFM/SEM images of a cilia lying on a glass substrate patterned with fiducial marks to allow revisitation of the same 10x10 micron area. The AFM images were taken in air. AFM imaging, the sample was coated with 10 nm of Au before imaging with scanning EM. The images are taken of the same cilium. As in figure 2, there is a distinctive tapered end (the distal end) and a more abrupt flat end on the basal side indicated in both images. The shape on the basal side of the cilium is most probably a function of the cleaving mechanism during isolation.

Structure, Dynamics and Forces of Cilia – EM, Fluorescence Microscopy and AFM

The figure above shows AFM data of a section of the length of a cilium imaged in buffer solution on a glass substrate. This cilium was isolated by dibucaine treatment of the epithelial cell culture and subsequent demembranation through exposure to triton. Along the ridge of the cilium, there are striations indicated here by the two black arrows. The distance between these striations is roughly 70nm which agrees well with what is expected for the inter-doublet distance in the axoneme. Direct AFM imaging of the axonemal skeleton is an exciting first step toward the goal of direct imaging of the dynein-microtubule doublet complex in a biologically relevant environment. Please visit the AFM Simulator poster for more information about this software.

Correlating AFM Data with Simulated AFM Data

Simulated AFM Data

5 m 25 m

AFM image of intact cilium AFM image of disrupted cilia

Cartoon of lung wall SEM image of cilia Cartoon of ciliary cross section

We will correlate the molecular resolution of TEM, AFM’s nanoscale surface mapping and liquid imaging capabilities, and the protein specificity of fluorescence microscopy to uncover the relationship between dynein motor activity and ciliary dynamics. We will analyze the same cilium substructures with all three techniques (fluorescence/optical, AFM, TEM), enabling substructure to be analyzed through mutually reinforcing data sets. We will correlate the molecular resolution of TEM, AFM’s nanoscale surface mapping and liquid imaging capabilities, and the protein specificity of fluorescence microscopy. We will analyze the same cilium substructures with all three techniques (fluorescence/optical, AFM, TEM), enabling substructure to be analyzed through mutually reinforcing data sets.

Cilia

Epithelial Cells

Mucus

AFMFluorescenceSEM / TEM

STRUCTURE

AFMFluorescence

DynamicsAFM

Forces

Preliminary Work

Pre-stressed Microtubule Doublets? The upper panels to the left show demembranated ciliary fragments imaged with AFM in liquid. The cilia are exfoliated and individual microtubule doublets lay on the surface in varying degrees of dissociation from their respective axonemes. Note that the individual microtubule doublets have a fairly consistent curvature. Given the known bending rigidity of microtubules, this degree of curvature is not expected without some asymmetry in the stress profile of the microtubule. This is suggestive of an asymmetry in the circumferential distribution of proteins on the microtubule doublets. This pre-stressing, if present, could have a significant role in the asymmetric beat mechanics of the cilium.

AFM (air)

AFM (liquid)

AFM (liquid)

AFM (liquid)

Simultaneous AFM/Fluorescenc imaging (Fibrin Fibers).

Successful Immunostaining of

epithelial cilia

http://www.bmb.psu.edu/courses/bisci004a/cells/

(liquid)

AFM (liquid)