Karl J. Aufderheide

Delineating the core regulatory elements crucial for directed cell migration by examining folic-acid-mediated responses

Summary Dictyostelium discoideum shows chemotaxis towards folic acid (FA) throughout vegetative growth, and towards cAMP during development. We determined the spatiotemporal localization of cytoskeletal and signaling molecules and investigated the FA-mediated responses in a number of signaling mutants to further our understanding of the core regulatory elements that are crucial for cell migration. Proteins enriched in the pseudopods during chemotaxis also relocalize transiently to the plasma membrane during uniform FA stimulation. In contrast, proteins that are absent from the pseudopods during migration redistribute transiently from the PM to the cytosol when cells are globally stimulated with FA. These chemotactic responses to FA were also examined in cells lacking the GTPases Ras C and G. Although Ras and phosphoinositide 3-kinase activity were significantly decreased in Ras G and Ras C/G nulls, these mutants still migrated towards FA, indicating that other pathways must support FA-mediated chemotaxis. We also examined the spatial movements of PTEN in response to uniform FA and cAMP stimulation in phospholipase C (PLC) null cells. The lack of PLC strongly influences the localization of PTEN in response to FA, but not cAMP. In addition, we compared the gradient-sensing behavior of polarized cells migrating towards cAMP to that of unpolarized cells migrating towards FA. The majority of polarized cells make U-turns when the cAMP gradient is switched from the front of the cell to the rear. Conversely, unpolarized cells immediately extend pseudopods towards the new FA source. We also observed that plasma membrane phosphatidylinositol 3,4,5-trisphosphate [PtdIns(3,4,5)P3] levels oscillate in unpolarized cells treated with Latrunculin-A, whereas polarized cells had stable plasma membrane PtdIns(3,4,5)P3 responses toward the chemoattractant gradient source. Results were similar for cells that were starved for 4 hours, with a mixture of polarized and unpolarized cells responding to cAMP. Taken together, these findings suggest that similar components control gradient sensing during FA- and cAMP-mediated motility, but the response of polarized cells is more stable, which ultimately helps maintain their directionality.

Clonal aging in Paramecium tetraurelia, absence of evidence for a cytoplasmic factor

Cells of Paramecium tetraurelia show clonal aging with characteristics comparable to the aging seen in tissue culture cell lines. An investigation is underway to determine the relative contributions, if any, of the cytoplasm and the macronucleus to the onset of senescence. Using a microinjection protocol, the importance of the cytoplasm to aging was tested. Cytoplasm was transferred from young cells into old cells to see if the mean age of death of the injected cell lines could be increased, compared with uninjected controls and sham injected controls. Cytoplasm from old cells was transferred into young cells to see if the mean age of death of the injected cell lines could be decreased, compared with sham injected and uninjected controls. In neither case was there any statistically significant change in mean ages of death. Furthermore, no changes can be demonstrated even with a protocol which transplanted 3 times the normal cytoplasmic volume. Thus, a cytoplasmic effect upon vegetative aging in P. tetraurelia cannot be demonstrated. Tests of a macronuclear influence are now underway.

A Microfluidic-Enabled Mechanical Microcompressor for the Immobilization of Live Single- and Multi-Cellular Specimens

A microcompressor is a precision mechanical device that flattens and immobilizes living cells and small organisms for optical microscopy, allowing enhanced visualization of sub-cellular structures and organelles. We have developed an easily fabricated device, which can be equipped with microfluidics, permitting the addition of media or chemicals during observation. This device can be used on both upright and inverted microscopes. The apparatus permits micrometer precision flattening for nondestructive immobilization of specimens as small as a bacterium, while also accommodating larger specimens, such as Caenorhabditis elegans, for long-term observations. The compressor mount is removable and allows easy specimen addition and recovery for later observation. Several customized specimen beds can be incorporated into the base. To demonstrate the capabilities of the device, we have imaged numerous cellular events in several protozoan species, in yeast cells, and in Drosophila melanogaster embryos. We have been able to document previously unreported events, and also perform photobleaching experiments, in conjugating Tetrahymena thermophila.

Directed Positioning of Micronuclei in Paramecium tetraurelia with Laser Tweezers: Absence of Detectible Damage After Manipulation

Possible covert damage from the use of the laser optical force trap (laser tweezers) to reposition micronuclei in Paramecium tetraurelia was assessed by measuring proliferation rates and postautogamous survival and mutation rates of cells after laser manipulations. No differences in subsequent daily proliferation rates among laser manipulated and various control classes of cells were seen. Similarly, the rates of postautogamous lethality and of “slow growth mutations” after repositioning of both micronuclei were not different from such rates in unmanipulated controls. In spite of extensive manipulations of micronuclei by the laser tweezers, there is no evidence of any damage induced by these manipulations. The laser tweezers therefore appears to be a tool of benign effect upon living cells, with tremendous potential use in many cell and developmental biological investigations.

Phenotypes associated with early clonal death in Paramecium tetraurelia

The aging characteristics of various known mutations of Paramecium tetraurelia have been studied. Among the mutations which had been previously isolated on the basis of the inability of the cell to do exocytosis of its trichocysts, one group was identified which had clonal life spans which were indistinguishable from, or were somewhat shorter than, the life span of wild-type. Another group of trichocyst mutations, however, had clonal life spans which were 1/3 to 1/4 that of wild-type. Further study of the phenotypes of these various mutations revealed that members of the short-lived group all had a common phenotypic defect: a frequent inability to properly divide the macronucleus during cell division. Another mutation which has normal trichocysts, but which shows macronuclear division defects, also has an extremely short life span. Thus, the short clonal life spans seen are not associated with the inability to do trichocyst exocytosis but rather are associated with the inability to properly divide the macronucleus. An hypothesis is presented which relates macronuclear misdivision to the short clonal life spans of the mutants expressing it. By extension of the hypothesis, the significance of normal macronuclear divisions to the normal aging process in P. tetraurelia is proposed.

ANALYSES OF INVERTED CILIARY ROWS IN PARAMECIUM. COMBINED LIGHT AND ELECTRON MICROSCOPIC OBSERVATIONS

Summary Many visible cytoskeletal and membranous differentiations organized around the basal bodies of ciliate protozoa reveal asymmetry (left-right) and/or polarity (anteriorposterior) in their organization. These cortical differentiations serve as orientation markers in analyses of cellular patterning and morphogenesis. We performed an integrated light and transmission electron microscopic analysis of 180° rotated (inverted) regions of the cortex of Paramecium . The internal organization of cortical units, and the ciliary rows they form, is unaffected by the inversion. Nevertheless, cortical structures in an inverted region show a reorientation (with respect to cellular axes) consistent with a planar 180° rotation of the affected ciliary rows. Typically-oriented regions adjacent to the inversion are unaffected by the inversion. A cell with an inversion displays two boundaries (junctures), with characteristic morphology, between normally-oriented cortex and the inversion. Therefore, the inversion is expressed as a reorientation of the affected cortical units relative to the cells orthogonal axes, but does not disrupt the internal organization of those cortical units. Cortical inversions are clonally stable during continued asexual propagation, and also through sexual exchanges. These observations demonstrated that significant differences in cellular phenotype can be sustained in cell lines in the absence of genotypic differences.

IFT57 stabilizes assembled intraflagellar transport complex and mediates transport of motility-related flagellar cargo.

ABSTRACT Intraflagellar transport (IFT) is essential for the assembly and maintenance of flagella and cilia. Recent biochemical studies have shown that IFT complex B (IFT-B) is comprised of two subcomplexes, IFT-B1 and IFT-B2. The IFT-B2 subunit IFT57 lies at the interface between IFT-B1 and IFT-B2. Here, using a Chlamydomonas reinhardtii mutant for IFT57, we tested whether IFT57 is required for IFT-B complex assembly by bridging IFT-B1 and IFT-B2 together. In the ift57-1 mutant, levels of IFT57 and other IFT-B proteins were greatly reduced at the whole-cell level. However, strikingly, in the protease-free flagellar compartment, while the level of IFT57 was reduced, the levels of other IFT particle proteins were not concomitantly reduced but were present at the wild-type level. The IFT movement of the IFT57-deficient IFT particles was also unchanged. Moreover, IFT57 depletion disrupted the flagellar waveform, leading to cell swimming defects. Analysis of the mutant flagellar protein composition showed that certain axonemal proteins were altered. Taken together, these findings suggest that IFT57 does not play an essential structural role in the IFT particle complex but rather functions to prevent it from degradation. Additionally, IFT57 is involved in transporting specific motility-related proteins. Highlighted Article: Intraflagellar transport (IFT) protein IFT57 maintains the pool size of IFT particles for ciliogenesis by stabilizing assembled IFT particle complexes, and is critical for flagellar waveform establishment.

Trichocyst phenotype transformation induced by macronuclear transplantation in Paramecium tetraurelia

A portion of the macronucleus of wild-type cells of Paramecium tetraurelia was removed and was injected into cells homozygous for the ftA mutation. The ftA mutants make defective trichocysts and are unable to perform normal trichocyst exocytosis. After injection, approx. 30% of the surviving cells show a phenotype shift from mutant to wild-type. This shift is stable during subsequent vegetative growth until clonal death. If, however, the hybrid cell lines are brought to autogamy (which discards the existing macronucleus and forms a new one from sexual products derived from a micronucleus), then the lines revert to the ftA phenotype. Since micronuclei were not transplanted, the phenotypic reversion after autogamy is to be expected, and demonstrates that the transformation affects the macronucleus only. A second series of injections involved transfer of a portion of the macronucleus from cells homozygous for the trichocyst ptA mutation into ftA host cells. These two mutations are genetically complementary, so the injection should be genetically equivalent to forming a double heterozygote. Approx. 20% of the injection survivors shift to wild-type. This shift is also vegetatively stable unless autogamy occurs; after autogamy, reversion to the ftA phenotype is seen. These results show that a portion of a macronucleus can be successfully transplanted from one cell to another and that, in the host cytoplasmic environment, normal gene expression and replication of a transplanted macronucleus does occur. The technique of macronuclear transplantation is significant to studies of the macronuclear contribution to clonal aging, and to studies on genetic control over trichocyst development.

A mechanical microcompressor for high resolution imaging of motile specimens

In order to obtain fine details in 3 dimensions (3D) over time, it is critical for motile biological specimens to be appropriately immobilized. Of the many immobilization options available, the mechanical microcompressor offers many benefits. Our device, previously described, achieves gentle flattening of a cell, allowing us to image finely detailed structures of numerous organelles and physiological processes in living cells. We have imaged protozoa and other small metazoans using differential interference contrast (DIC) microscopy, orientation-independent (OI) DIC, and real-time birefringence imaging using a video-enhanced polychromatic polscope. We also describe an enhancement of our previous design by engineering a new device where the coverslip mount is fashioned onto the top of the base; so the entire apparatus is accessible on top of the stage. The new location allows for easier manipulation of the mount when compressing or releasing a specimen on an inverted microscope. Using this improved design, we imaged immobilized bacteria, yeast, paramecia, and nematode worms and obtained an unprecedented view of cell and specimen details. A variety of microscopic techniques were used to obtain high resolution images of static and dynamic cellular and physiological events.

Migration of Dictyostelium discoideum to the Chemoattractant Folic Acid

Dictyostelium discoideum can be grown axenically in a cultured media or in the presence of a natural food source, such as the bacterium Klebsiella aerogenes (KA). Here we describe the advantages and methods for growing D. discoideum on a bacterial lawn for several processes studied using this model system. When grown on a bacterial lawn, D. discoideum show positive chemotaxis towards folic acid (FA). While these vegetative cells are highly unpolarized, it has been shown that the signaling and cytoskeletal molecules regulating the directed migration of these cells are homologous to those seen in the motility of polarized cells in response to the chemoattractant cyclic adenosine monophosphate (cAMP). Growing D. discoideum on KA stimulates chemotactic responsiveness to FA. A major advantage of performing FA-mediated chemotaxis is that it does not require expression of the cAMP developmental program and therefore has the potential to identify mutants that are purely unresponsive to chemoattractant gradients. The cAMP-mediated chemotaxis can appear to fail when cells are developmentally delayed or do not up-regulate genes needed for cAMP-mediated migration. In addition to providing robust chemotaxis to FA, cells grown on bacterial lawns are highly resistant to light damage during fluorescence microscopy. This resistance to light damage could be exploited to better understand other biological processes such as phagocytosis or cytokinesis. The cell cycle is also shortened when cells are grown in the presence of KA, so the chances of seeing a mitotic event increases.