Karl A. Johnson

Oscillatory Flows Induced by Microorganisms Swimming in Two Dimensions

We present the first time-resolved measurements of the oscillatory velocity field induced by swimming unicellular microorganisms. Confinement of the green alga C. reinhardtii in stabilized thin liquid films allows simultaneous tracking of cells and tracer particles. The measured velocity field reveals complex time-dependent flow structures, and scales inversely with distance. The instantaneous mechanical power generated by the cells is measured from the velocity fields and peaks at 15 fW. The dissipation per cycle is more than 4 times what steady swimming would require.

Enhancement of biomixing by swimming algal cells in two-dimensional films.

Fluid mixing in active suspensions of microorganisms is important to ecological phenomena and presents a fascinating stochastic process. We investigate the mixing produced by swimming unicellular algal cells (Chlamydomonas) in quasi-two-dimensional liquid films by simultaneously tracking the motion of the cells and that of microscopic passive tracer particles advected by the fluid. The reduced spatial dimension of the system leads to long-range flows and a surprisingly strong dependence of tracer transport on the concentration of swimmers, which is explored over a wide range. The mean square displacements are well described by a stochastic Langevin model, which is used to parameterize the mixing. The effective diffusion coefficient D grows rapidly with the swimmer concentration Φ as D ∼ Φ3/2, as a result of the increasing frequency of tracer-swimmer interactions and the long-range hydrodynamic disturbances created by the swimmers. Conditional sampling of the tracer data based on the instantaneous swimmer position shows that the rapid growth of the diffusivity enhancement with concentration must be due to particle interactions with multiple swimmers simultaneously. Finally, the anomalous probability distributions of tracer displacements become Gaussian at high concentration, but manifest strong power-law tails at low concentration, while the tracer displacements always grow diffusively in time.

Self Assembly of Coiled-Coil Peptide−Porphyrin Complexes

We are interested in the controlled assembly of photoelectronic materials using peptides as scaffolds and porphyrins as the conducting material. We describe the integration of a peptide-based polymer strategy with the ability of designed basic peptides to bind anionic porphyrins in order to create regulated photoelectronically active biomaterials. We have described our peptide system in earlier work, which demonstrates the ability of a peptide to form filamentous materials made up of self-assembling coiled-coil structures. We have modified this peptide system to include lysine residues appropriately positioned to specifically bind meso-tetrakis(4-sulfonatophenyl)porphine (TPPS(4)), a porphyrin that contains four negatively charged sulfonate groups at neutral pH. We measure the binding of TPPS(4) to our peptide using UV--visible and fluorescence spectroscopies to follow the porphyrin signature. We determine the concomitant acquisition of helical secondary structure in the peptide upon TPPS(4) binding using circular dichroism spectropolarimetry. This binding fosters polymerization of the peptide, as shown by absorbance extinction effects in the peptide CD spectra. The morphologies of the peptide/porphyrin complexes, as imaged by atomic force microscopy, are consistent with the coiled-coil polymers that we had characterized earlier, except that the heights are slightly higher, consistent with porphyrin binding. Evidence for exciton coupling in the copolymers is shown by red-shifting in the UV--visible data, however, the coupling is weak based on a lack of fluorescence quenching in fluorescence experiments.

Basal bodies and DNA

The possibility that basal bodies/centrioles contain nucleic acid has been a controversial topic in cell biology for several decades. These structures are conservatively replicated, are segregated at mitosis, and play a prominent role in cytoskeletal organization; thus, some have chosen to view centrioles as autonomous, self-replicating entities, and have searched for centriole-associated DNA. Two years ago, a report suggested that a chromosome defined by a specific linkage group is located within each basal body of the unicellular green alga Chlamydomonas. Several recent investigations have presented new data that force a re-evaluation of that conclusion.

Replication of basal bodies and centrioles

During the past year, studies on the centrioles and basal bodies of animal and algal cells, and the spindle pole bodies of yeast and other fungi, have added significantly to our knowledge of how these cell organelles form and how they function in initiating microtubule assembly throughout the cell cycle. Most of these studies have used antibodies to identify proteins within and around these organelles and, in some cases, to disrupt their ability to nucleate microtubules. Genetic methods have been used to identify specific proteins, including a new member of the tubulin superfamily, involved in the function and replication of spindle pole bodies and centrioles.

Characterization of a Molecular Chaperone Present in the Eukaryotic Flagellum

ABSTRACT Chlamydomonas flagella contain a molecular chaperone now identified as HSP70A, a major cytoplasmic isoform. HSP70A synthesis is upregulated by deflagellation, and its distribution in the flagellum overlaps with the IFT kinesin-II motor FLA10. HSP70A may chaperone flagellar proteins during transport, participating in the assembly and maintenance of the flagellum.

Effect of Helical Flanking Sequences on the Morphology of Polyglutamine-Containing Fibrils

A peptide model system has been developed to study the effects of helical flanking sequences on polyglutamine aggregation. In a companion manuscript, the kinetics of aggregation are described, comparing the influence of a well-defined heterotetrameric coiled coil to that of the helix-rich structure found in Htt(NT), a 17-residue flanking sequence found in the huntingtin protein, on polyglutamine aggregation. Here, the morphological characterization of the resultant fibrils that form for a set of peptides is reported, only one of which, KKQ25KK, has been previously studied. A careful analysis of TEM and AFM images of KKQ25KK confirms that it forms bundled fibrils of varying length and reveals, unexpectedly, that they are composed of fully extended cross-β-strands. Second, it is shown that helical flanking sequences do not disrupt the assembly of a core cross-β-sheet structure, but such flanking sequences can influence higher order processes, such as inhibiting the bundling of the fibrils.

Chapter 23 Immunoelectron Microscopy

Publisher Summary The purpose of this chapter is to provide a practical introduction to the technique of immunoelectron microscopy that is a tool for the ultrastructural localization of flagellar/ciliary components. As polyclonal antibodies recognize multiple antigenic determinants per target molecule, they generally produce better signals than monoclonal antibodies. Antibody specificity is to be evaluated by immunoblot analysis. There is detailing of the specimen preparation involving embedding and thin sectioning, embedment of Chlamydomonas in L.R. white, flagellar whole mounts, and tips for incubating EM grids on various solutions. Frozen thin sections provide good membrane preservation useful for the analysis of flagellar membrane-associated antigens. Freed from the masking effects of heavy fixation, dehydration, and embedment, axonemal whole mounts show excellent labeling densities. There is also a description of the method for the preparation of Chlamydomonas axonemal whole mounts. There are tips for incubating EM grids on various solutions. Successful immunolabeling of material on EM grids requires that the same grid be handled many times without damage. One way to accomplish this is to use a modified inoculating loop to move grids from solution-to-solution. Also a description of immunolabeling technique has been provided. Details have been presented on the choice of gold marker and Immunogold labeling of specimens on EM grids. In the absence of poststaining, the gold particles stand out well, to evaluate levels of specific staining of structures, and of background labeling. The initial screening of antibodies by immunofluorescence indicates where antigen should be detected. An ideal control is provided by performing identical localizations on mutant cells that lack the specific gene product being localized. In evaluation of antisera in immunofluorescence, a positive signal provides a guide, indicating where antigen is localized and suggesting that the antigen is concentrated enough to be detected at the EM level.

Genomic amplification in the cotyledon parenchyma of common bean

The cotyledon parenchyma cells of common bean (Phaseolus vulgaris L.) produce large quantities of storage proteins during embryo maturation; throughout this period, these cells also accumulate nuclear DNA (nDNA). To investigate the basis of this nDNA accumulation, we have measured storage protein mRNA pools, nDNA mass, and gene copy number at specific stages of cotyledon development. RNA blotting and hybridization show that transcripts encoding the major embryospecific storage proteins are present very early in cotyledon development, accumulate in coordinate fashion to peak during mid-maturation, and fall in abundance prior to the onset of dormancy. During this same period, nDNA mass per parenchyma cell nucleus, as measured by Feulgen microspectrophotometry, increases from 2C–4C to about 64C (C being the haploid germ cell genomic complement). The nDNA values do not cluster around integral multiples of the diploid 2C amount. DNA blotting and hybridization are used to evaluate the relative representations of different classes of the bean genome in DNA samples isolated from vegetative tissues, from cotyledons beginning to accumulate storage proteins, and from cotyledons of late maturation embryos entering dormancy. The results demonstrate that the observed DNA accumulation in the cotyledon parenchyma is due to overlapping rounds of replication of the complete genome and not to disproportionate amplification of specific sequences nor to random DNA synthesis.

Measuring oscillatory velocity fields due to swimming algae

In this fluid dynamics video, we present the first time-resolved measurements of the oscillatory velocity field induced by swimming unicellular microorganisms. Confinement of the green alga C. reinhardtii in stabilized thin liquid films allows simultaneous tracking of cells and tracer particles. The measured velocity field reveals complex time-dependent flow structures, and scales inversely with distance. The instantaneous mechanical power generated by the cells is measured from the velocity fields and peaks at 15 fW. The dissipation per cycle is more than four times what steady swimming would require.