Immunofluorescent localization of microtubules, microfilaments and DNA in human lung cancer cells. Note the microtubule cytoskeleton in a growing cell has a single focusn near the nucleus (left);
when the cell starts to divide, the interphase microtubules disassemble and are replaced by a bipolar mitotic spindle (right).

Structural
Biology
@
Haverford

Karl Johnson

In our lab we seek to understand how biological systems assemble and maintain a diversity of structural systems such as the cytoskeleton, the Eukaryoticic flagellum and virus particles. Our interests range from basic scientific questions to applications in medicine and nanotechnology.

The cytoskeleton: Eukaryoticic cells assemble internal architectures of microtubules, microfilaments and intermediate filaments that help structure cells and tissues. These complementary elements can be dynamic as well as static and can bear both loads and tension. They help organize the growing interphase cell and are reconfigured to construct the mitotic apparatus as a cell prepares to divide (Figure 1). Biological motor proteins such as dyneins, kinesins and myosins use cytoskeletal polymers as tracks, delivering a variety of cargo throughout cells and animating the mitotic spindle. Proper assembly, maintenance and turnover of the cytoskeleton are important for normal cellular activities, and defects in cytoskeletal components are responsible for a wide spectrum of human diseases including the progression to cancer.

The Eukaryoticic flagellum: The Eukaryoticic flagellum is an exquisite biological nanomachine constructed of cytoskeletal components. Present on the cells of lower plants, algae and animals, flagella are long, thread-like organelles that consist of a structural core, the “9+2” microtubule axoneme, surrounded by a specialized extension of the cell membrane. Flagellar beating is driven by axonemal dynein motor proteins, coordinated by the central pair apparatus. Different patterns of regulation result in a variety of waveforms from a symmetric pushing motion to an asymmetric "wave" that moves a cell relative to its fluid surroundings.

Our two decades of experience exploring flagella in the green alga Chlamydomonas have revealed novel insights into how cells assemble, regulate and maintain flagella. As a postdoctoral fellow in the laboratory of Joel Rosenbaum (Yale University), I showed that unassembled axonemal proteins are directed from the cytoplasm of the cell body, where they are synthesized on ribosomes, to the distal ends of flagella, where they are assembled into the elongating axoneme (Johnson and Rosenbaum, 1992). This work led to the observation of a novel transport mechanism, which we named IntraFlagellar Transport (IFT) (Kozminski et al., 1993), that shuttles “rafts” of material along the axonemal outer doublet microtubules using kinesin II and cytoplasmic dynein motors. Defects in IFT are now understood to underlie a wide variety of human diseases ranging from retinal degeneration to polycystic kidney disease, generating a renaissance of interest in basic flagellar biology (recently reviewed by (Pedersen et al., 2006; Qin et al., 2004)).

Tubulin localization in the
biflagellate green alga Chlamydomonas

 

Work from our lab at Haverford discovered (Bloch and Johnson, 1995) and characterized (Shapiro et al., 2005) a member of the Hsp70 family of molecular chaperones in the flagellum, where its distribution overlaps with the anterograde IFT kinesin FLA10 (Figure 4). We have proposed that this chaperone, called HSP70A, may accompany newly synthesized axonemal proteins during transport into the flagellar compartment and to the tip assembly site. Flagellar HSP70A may also participate in the maintenance of assembled axonemal proteins, refolding and repairing them in situ to keep the flagellar machine functioning. Others have added evidence that HSP70A also localizes to an ATP-regenerating complex located on the central pair apparatus, helping power this nanomachine (Mitchell et al., 2005). We are currently exploring further the flagellar functions of HSP70A using RNAi approaches (Rohr et al., 2004) and expression of molecularly-engineered dominant negative constructs.

Image left: Colocalization of the molecular chaperone HSP70A and the IFT kinesin motor FLA10 in Chlamydomonas flagella (from Shapiro, Ingram and Johnson, 2005)

We are grateful for support for our work in Chlamydomonas provided by the National Science Foundation (MCB-9506236 and MCB-9982733). "Any opinions, findings and conclusions or recommendations expressed in this material are those of the author(s) and do not necessarily reflect the views of the National Science Foundation (NSF)."

 

Recently, we entered into a collaboration with Jerry Gollub in the Haverford Department of Physics to understand better the biophysics of Chlamydomonas cell swimming. Using a special device designed to stretch thin liquid films across the field of an optical microscope, we have recorded high-speed video sequences of cells swimming in 2D environments. By incorporating small spheres into films with the cells, we have mapped the time-resolved velocity field around the swimming cell (Guasto, Johnson and Gollub, 2010). While cells appear to make smooth forward progress when observed at 50 frames per second (fps), imaging at 500 fps reveals the oscillatory nature of swimming, with cells breast-stroking forward with each power stoke but then moving backward with each recovery stroke. This oscillatory movement requires that swimming cells expend several-fold greater energy than previously thought, generating a peak mechanical power of 15 femtowatts (fW). While the "big step forward, small step back" might seem "wasteful," it is necessary for cells to make forward progress in a low Reynolds Number environment (a condition in which viscous fluid conditions predominate). We have also studied how cells' swimming enhances the mixing at the nanoscale, enhancing diffusion at higher cell densities (Kurtuldu, Guasto, Johnson and Gollub 2011). Th results of our collaborations have implications for understanding the work done by flagella and cilia in many environments, including the human trachea, kidneys and reproductive tract.

Time-resolved map of the velocity field around a swimming Chlamydomonas cell

Bacterial viruses (bacteriophage): As part of a nanotechnology working group at Haverford, initially supported by the David and Lucille Packard Foundation, we became interested in 2004 in using biological systems as inspiration for the design of novel, self-assembling nanoscale architectures. Using M13, a filamentous virus that infects E. coli, we have initiated an ambitious project to explore phages as a component for the development of novel structures and machines on the nanometer scale. M13 is well-known to the first generation of molecular biologists as a system for the isolation of single stranded DNA for sequencing reactions (Messing, 1991). Each M13 virus particle is a long filament 900 nm in length but only 8 nm in width and 1012 virus particles can be isolated from a milliliter of an overnight bacterial culture. Each phage consists of a single-stranded 6.4Kb DNA core, ensheathed in a coat of 2700 copies of a short 50 amino acid peptide called pVIII. One end of the virus ends in 5 copies each of the minor coat proteins VII and IX, while the other bears 5 copies each of pIII and pVI. The filamentous bacteriophage M13 (and its close relative fd) have also found utility in phage display, in which novel epitopes are expressed as fusions to specific coat proteins via molecular engineering (reviewed by (Kehoe and Kay, 2005; Sidhu, 2001; Smith and Petrenko, 1997)). Vectors are available for phage display on all five different coat proteins in either multivalent (all copies) or mixed valency (in which wild-type and display versions are co-expressed) configurations. Phage display has been used to screen libraries for specific affinity interactions or (in a reverse sense) to add defined epitope(s) to the surface of the phage particles.


Negative stained EM of M13 bacteriophage

We have started by making fluorescent phage, using chemistry to link fluorescent molecules to phage particles and are able to image and track individual virus particles. These photonic phage can absorb, transfer and reemit energy at several different wavelengths (depending upon the fluorochrome to which they have been conjugated). We are working to study the properties of these components, hoping to develop these modified phage as energy carrying "wires", solar cell components, and environmental probes. Other studies in the lab are directed at developing mechanisms to anchor these phage to specific surfaces and to assemble these phage into higher order structures. Come join us in our explorations of this new frontier!

 

We are grateful for support for our work on viral assembly and nanotechnology provided by the Packard Foundation and the Biomaterials Program in the Division of Materials Research at the National Science Foundation (DMR-0804944). "Any opinions, findings and conclusions or recommendations expressed in this material are those of the author(s) and do not necessarily reflect the views of the National Science Foundation (NSF)."

 

References cited:

Bloch, M.A., and Johnson K.A. 1995. Identification of a molecular chaperone in the Eukaryoticic flagellum and its localization to the site of microtubule assembly. J Cell Sci. 108 ( Pt 11):3541-5.
Guasto JS, Johnson KA, Gollub JP. 2010. Oscillatory flows induced by microorganisms swimming in two dimensions. Phys Rev Lett. 2010 Oct 15;105(16):168102.
Johnson, K.A., and J.L. Rosenbaum. 1992. Polarity of flagellar assembly in Chlamydomonas. J Cell Biol. 119:1605-11.
Kehoe, J.W., and B.K. Kay. 2005. Filamentous phage display in the new millennium. Chem Rev. 105:4056-72.
Kozminski, K.G., K.A. Johnson, P. Forscher, and J.L. Rosenbaum. 1993. A motility in the Eukaryoticic flagellum unrelated to flagellar beating. Proc Natl Acad Sci U S A. 90:5519-23.
Messing, J. 1991. Cloning in M13 phage or how to use biology at its best. Gene. 100:3-12.
Mitchell, B.F., L.B. Pedersen, M. Feely, J.L. Rosenbaum, and D.R. Mitchell. 2005. ATP production in Chlamydomonas reinhardtii flagella by glycolytic enzymes. Mol Biol Cell. 16:4509-18.
Pedersen, L.B., S. Geimer, and J.L. Rosenbaum. 2006. Dissecting the molecular mechanisms of intraflagellar transport in Chlamydomonas. Curr Biol. 16:450-9.
Qin, H., D.R. Diener, S. Geimer, D.G. Cole, and J.L. Rosenbaum. 2004. Intraflagellar transport (IFT) cargo: IFT transports flagellar precursors to the tip and turnover products to the cell body. J Cell Biol. 164:255-66.
Rohr, J., N. Sarkar, S. Balenger, B.R. Jeong, and H. Cerutti. 2004. Tandem inverted repeat system for selection of effective transgenic RNAi strains in Chlamydomonas. Plant J. 40:611-21.
Shapiro, J., J. Ingram, and K.A. Johnson. 2005. Characterization of a molecular chaperone present in the Eukaryoticic flagellum. Eukaryotic Cell. 4:1591-4.
Sidhu, S.S. 2001. Engineering M13 for phage display. Biomol Eng. 18:57-63.
Smith, G.P., and V.A. Petrenko. 1997. Phage display. Chem Rev. 97:391-410.

Selected recent publications from our lab and collaborations: (* denotes undergraduate co-authors)

Kurtuldu H, Guasto JS, Johnson KA, Gollub JP. (2011) Enhancement of biomixing by swimming algal cells in two-dimensional films. Proc Natl Acad Sci U S A. 2011 Jun 28;108(26):10391-5. Epub 2011 Jun 9. PMID: 21659630

Guasto JS, Johnson KA, Gollub JP. (2010) Oscillatory flows induced by microorganisms swimming in two dimensions. Phys Rev Lett. 105(16):168102. PMID: 21231018

Kokona B, *Kim AM, *Roden RC, *Daniels JP, *Pepe-Mooney BJ, *Kovaric BC, de Paula JC, Johnson KA, Fairman R. (2009) Self Assembly of Coiled-Coil Peptide-Porphyrin Complexes. Biomacromolecules. 2009 Apr 17. PMID: 19374349

*Shapiro J, *Ingram J, Johnson KA (2005) Characterization of a molecular chaperone present in the Eukaryoticic flagellum. Eukaryotic Cell. 4(9):1591-4. PMID: 16151252

*Rigotti DJ, Kokona B, *Horne T, *Acton EK, *Lederman CD, Johnson KA, Manning RS, Kane SA, Smith WF, Fairman R. (2005) Quantitative atomic force microscopy image analysis of unusual filaments formed by the Acanthamoeba castellanii myosin II rod domain. Anal Biochem. 346(2):189-200. PMID:16213459

   
Links:
   
Karl's Curriculum Vitae

Cell Architecture (HBio302)

Modeling the Organism (HBio200) The Haverford College Biology Department The Haverford College Home Page

 

This page last updated on January 23, 2011