Directed movement toward, translocation along, penetration into and exit from vascular networks by breast cancer cells in 3D.

We developed a computer-assisted platform using laser scanning confocal microscopy to 3D reconstruct in real-time interactions between metastatic breast cancer cells and human umbilical vein endothelial cells (HUVECs). We demonstrate that MB-231 cancer cells migrate toward HUVEC networks, facilitated by filopodia, migrate along the network surfaces, penetrate into and migrate within the HUVEC networks, exit and continue migrating along network surfaces. The system is highly amenable to 3D reconstruction and computational analyses, and assessments of the effects of potential anti-metastasis monoclonal antibodies and other drugs. We demonstrate that an anti-RHAMM antibody blocks filopodium formation and all of the behaviors that we found take place between MB-231 cells and HUVEC networks.

Candida albicans Double Mutants Lacking both EFG1 and WOR1 Can Still Switch to Opaque.

Candida albicans, a pervasive opportunistic pathogen, undergoes a unique phenotypic transition from a "white" phenotype to an "opaque" phenotype. The switch to opaque impacts gene expression, cell morphology, wall structure, metabolism, biofilm formation, mating, virulence, and colonization of the skin and gastrointestinal (GI) tract. Although the regulation of switching is complex, a paradigm has evolved from a number of studies, in which, in its simplest form, the transcription factors Efg1 and Wor1 play central roles. When EFG1 is upregulated under physiological conditions, it represses WOR1, an activator of white-to-opaque switching, and the cell expresses the white phenotype; when EFG1 is downregulated, WOR1 is derepressed and activates expression of the opaque phenotype. Deletion of either EFG1 or WOR1 supports this yin-yang model of regulation. Here, we demonstrate that this simple model is insufficient, since strains in which WOR1 and EFG1 are simultaneously deleted can still be induced to switch en masse from white to opaque. Opaque cells of double mutants (efg1-/- wor1-/- ) are enlarged and elongate, form an enlarged vacuole, upregulate mCherry under the control of an opaque-specific promoter, form opaque cell wall pimples, express the opaque phenotype in lower GI colonization, and, if MTL homozygous, form conjugation tubes in response to pheromone and mate. These results can be explained if the basic and simplified model is expanded to include a WOR1-independent alternative opaque pathway repressed by EFG1IMPORTANCE The switch from white to opaque in Candida albicans was discovered 33 years ago, but it is still unclear how it is regulated. A regulatory paradigm has emerged in which two transacting factors, Efg1 and Wor1, play central roles, Efg1 as a repressor of WOR1, which encodes an activator of the transition to the opaque phenotype. However, we show here that if both EFG1 and WOR1 are deleted simultaneously, bona fide opaque cells can still be induced en masse These results are not compatible with the simple paradigm, suggesting that an alternative opaque pathway (AOP) exists, which can activate expression of opaque and, like WOR1, is repressed by EFG1.

EFG1 Mutations, Phenotypic Switching, and Colonization by Clinical a/α Strains of Candida albicans.

The transcription factor EFG1 functions as a suppressor of white-to-opaque and white-to-gray switching in a/α strains of Candida albicans In a collection of 27 clinical isolates, 4 of the 17 EFG1/EFG1 strains, 1 of the 2 EFG1/efg1 strains, and all 8 of the efg1/efg1 strains underwent white-to-opaque switching. The four EFG1/EFG1 strains, the one EFG1/efg1 strain, and one of the eight efg1/efg1 strains that underwent switching to opaque did not switch to gray and could not be complemented with a copy of EFG1 Competition experiments in a mouse model for gastrointestinal (GI) colonization confirmed that efg1/efg1 cells rapidly outcompete EFG1/EFG1 cells, and in plating experiments, formed colonies containing both gray and opaque cells. Direct microscopic analysis of live cells in the feces, however, revealed that the great majority of cells were opaque, suggesting opaque, not gray, may be the dominant phenotype at the site of colonization.IMPORTANCE Close to half of a collection of 27 clinical a/α isolates of Candida albicans underwent white-to-opaque switching. Complementation experiments revealed that while approximately half of the a/α switchers were due to EFG1 mutations, the remaining half were due to mutations in other genes. In addition, the results of competition experiments in a mouse GI tract colonization model support previous observations that efg1/efg1 cells rapidly outcompete EFG1/EFG1 strains, but direct microscopic analysis reveals that the major colonizing cells were opaque, not gray.

Reciprocal signaling and direct physical interactions between fibroblasts and breast cancer cells in a 3D environment.

Tumorigenic cells undergo cell aggregation and aggregate coalescence in a 3D Matrigel environment. Here, we expanded this 3D platform to assess the interactions of normal human dermal fibroblasts (NHDFs) and human primary mammary fibroblasts (HPMFs) with breast cancer-derived, tumorigenic cells (MDA-MB-231). Medium conditioned by MDA-MB-231 cells activates both types of fibroblasts, imbuing them with the capacity to accelerate the rate of aggregation and coalescence of MDA-MB-231 cells more than four fold. Acceleration is achieved 1) by direct physical interactions with MDA-MB-231 cells, in which activated fibroblasts penetrate the MDA-MB-231/Matrigel 3D environment and function as supporting scaffolds for MDA-MB-231 aggregation and coalescence, and 2) through the release of soluble accelerating factors, including matrix metalloproteinase (MMPs) and, in the case of activated NHDFs, SDF-1α/CXCL12. Fibroblast activation includes changes in morphology, motility, and gene expression. Podoplanin (PDPN) and fibroblast activation protein (FAP) are upregulated by more than nine-fold in activated NHDFs while activated HPMFs upregulate FAP, vimentin, desmin, platelet derived growth factor receptor A and S100A4. Overexpression of PDPN, but not FAP, in NHDF cells in the absence of MDA-MB-231-conditioned medium, activates NHDFs. These results reveal that complex reciprocal signaling between fibroblasts and cancer cells, coupled with their physical interactions, occurs in a highly coordinated fashion that orchestrates aggregation and coalescence, behaviors specific to cancer cells in a 3D environment. These in vitro interactions may reflect events involved in early tumorigenesis, particularly in cases of field cancerization, and may represent a new mechanism whereby cancer-associated fibroblasts (CAFs) promote tumor growth.

Roles of the Transcription Factors Sfl2 and Efg1 in White-Opaque Switching in a/α Strains of Candida albicans.

Candida albicans remains the most pervasive fungal pathogen colonizing humans. The majority of isolates from hosts are heterozygous at the mating type locus (MTLa/α), and a third of these have recently been shown to be capable of switching to the opaque phenotype. Here we have investigated the roles of two transcription factors (TFs) Sfl2 and Efg1, in repressing switching in a/α strains. Deleting either gene results in the capacity of a/α cells to switch to opaque en masse under facilitating environmental conditions, which include N-acetylglucosamine (GlcNAc) as the carbon source, physiological temperature (37°C), and high CO2 (5%). These conditions are similar to those in the host. Our results further reveal that while glucose is a repressor of sfl2Δ and efg1Δ switching, GlcNAc is an inducer. Finally, we show that when GlcNAc is the carbon source, and the temperature is low (25°C), the efg1Δ mutants, but not the sfl2Δ mutants, form a tiny, elongate cell, which differentiates into an opaque cell when transferred to conditions optimal for a/α switching. These results demonstrate that at least two TFs, Sfl2 and Efg1, repress switching in a/α cells and that a/α strains with either an sfl2Δ or efg1Δ mutation can switch en masse but only under physiological conditions. The role of opaque a/α cells in commensalism and pathogenesis must, therefore, be investigated.IMPORTANCE More than 95% of Candida albicans strains isolated from humans are MTLa/α, and approximately a third of these can undergo the white-to-opaque transition. Therefore, besides being a requirement for MTL-homozygous strains to mate, the opaque phenotype very likely plays a role in the commensalism and pathogenesis of nonmating, a/α populations colonizing humans.

Candida albicans White-Opaque Switching Influences Virulence but Not Mating during Oropharyngeal Candidiasis.

The capacity of Candida albicans to switch reversibly between the white phenotype and the opaque phenotype is required for the fungus to mate. It also influences virulence during hematogenously disseminated candidiasis. We investigated the roles of the mating type loci (MTL) and white-opaque switching in the capacity of C. albicans to mate in the oropharynx and cause oropharyngeal candidiasis (OPC). When immunosuppressed mice were orally infected with mating-competent opaque a/a and α/α cells either alone or mixed with white cells, no detectable mating occurred, indicating that the mating frequency was less than 1.6 × 10-6 Opaque cells were also highly attenuated in virulence; they either were cleared from the oropharynx or switched to the white phenotype during OPC. Although there were strain-to-strain differences in the virulence of white cells, they were consistently more virulent than opaque cells. In vitro studies indicated that relative to white cells, opaque cells had decreased capacity to invade and damage oral epithelial cells. The reduced invasion of at least one opaque strain was due to reduced surface expression of the Als3 invasin and inability to activate the epidermal growth factor receptor, which is required to stimulate the epithelial cell endocytic machinery. These results suggest that mating is a rare event during OPC because opaque cells have reduced capacity to invade and damage the epithelial cells of the oral mucosa.

Generating a Battery of Monoclonal Antibodies Against Firefly Luciferase for Dot Blot Analysis, Western Blot Analysis, and Immunostaining of Cells in Culture and Paraffin Sections.

Firefly luciferase (FLuc) is commonly used as a reporter gene PpyLuc1 in bioanalytical assays. We have produced five mouse-derived monoclonal antibodies (mAbs) that recognize FLuc. The mAbs, DSHB-LUC-2, DSHB-LUC-3, DSHB-LUC-9, DSHB-LUC-16, and DSHB-LUC-24, were generated by immunizing mice with purified 6xHIS-tagged FLuc (6xHis-FLuc) in suspension with an adjuvant. All five were validated by dot blots. Four of the mAbs provided strong signals in western blot analysis, and one a weak signal. All five were validated for immunostaining in fixed cell culture. Only one stained cells embedded in paraffin. The five mAbs are available at cost through the Developmental Studies Hybridoma Bank (DSHB), a nonprofit National Resource created by the National Institutes of Health.

Protocol for Identifying Natural Agents That Selectively Affect Adhesion, Thickness, Architecture, Cellular Phenotypes, Extracellular Matrix, and Human White Blood Cell Impenetrability of Candida albicans Biofilms.

In the screening of natural plant extracts for antifungal activity, assessment of their effects on the growth of cells in suspension or in the wells of microtiter plates is expedient. However, microorganisms, including Candida albicans, grow in nature as biofilms, which are organized cellular communities with a complex architecture capable of conditioning their microenvironment, communicating, and excluding low- and high-molecular-weight molecules and white blood cells. Here, a confocal laser scanning microscopy (CLSM) protocol for testing the effects of large numbers of agents on biofilm development is described. The protocol assessed nine parameters from a single z-stack series of CLSM scans for each individual biofilm analyzed. The parameters included adhesion, thickness, formation of a basal yeast cell polylayer, hypha formation, the vertical orientation of hyphae, the hyphal bend point, pseudohypha formation, calcofluor white staining of the extracellular matrix (ECM), and human white blood cell impenetrability. The protocol was applied first to five plant extracts and derivative compounds and then to a collection of 88 previously untested plant extracts. They were found to cause a variety of phenotypic profiles, as was the case for 64 of the 88 extracts (73%). Half of the 46 extracts that did not affect biofilm thickness affected other biofilm parameters. Correlations between specific effects were revealed. The protocol will be useful not only in the screening of chemical libraries but also in the analysis of compounds with known effects and mutations.

Binding Sites in the EFG1 Promoter for Transcription Factors in a Proposed Regulatory Network: A Functional Analysis in the White and Opaque Phases of Candida albicans.

In Candida albicans the transcription factor Efg1, which is differentially expressed in the white phase of the white-opaque transition, is essential for expression of the white phenotype. It is one of six transcription factors included in a proposed interactive transcription network regulating white-opaque switching and maintenance of the alternative phenotypes. Ten sites were identified in the EFG1 promoter that differentially bind one or more of the network transcription factors in the white and/or opaque phase. To explore the functionality of these binding sites in the differential expression of EFG1, we generated targeted deletions of each of the 10 binding sites, combinatorial deletions, and regional deletions using a Renilla reniformis luciferase reporter system. Individually targeted deletion of only four of the 10 sites had minor effects consistent with differential expression of EFG1, and only in the opaque phase. Alternative explanations are considered.

Generation and Validation of Monoclonal Antibodies Against the Maltose Binding Protein.

The maltose binding protein (MBP) is a commonly used protein tag. Two monoclonal antibodies (mAbs) were generated against the MBP by immunizing mice with purified 6xHis-tagged MBP (6xHis-MBP). A nontoxic adjuvant cocktail of poly(I:C) and anti-CD40 mAb was used. The two mAbs, 3D7 and 2A1, are demonstrated to be effective in immunoprecipitation, immunoblotting, western blot hybridization, and the ELISA assay. These two mAbs are available individually or in combination at cost through the Developmental Studies Hybridoma Bank, a nonprofit National Resource created by the National Institutes of Health.

Role of Tec1 in the development, architecture, and integrity of sexual biofilms of Candida albicans.

MTL-homozygous ( A: / A: or α/α) white cells form a complex sexual biofilm that exhibits the same architecture as that of MTL-heterozygous ( A: /α) pathogenic biofilms. However, the former is regulated by the mitogen-activated protein (MAP) kinase pathway, while the latter is regulated by the Ras1/cyclic AMP (cAMP) pathway. We previously demonstrated that in the formation of an MTL-homozygous, mature (48 h) sexual biofilm in RPMI 1640 medium, the MAP kinase pathway targets Tec1 rather than Cph1, the latter of which is the target of the same pathway, but for the opaque cell mating response. Here we continued our analysis of the role of Tec1 by comparing the effects of deleting TEC1 on initial adhesion to silicone elastomer, high-resolution confocal microscopy assessments of the stages and cellular phenotypes during the 48 h of biofilm development, human white cell penetration, and biofilm fragility. We show that although Tec1 plays only a minor role in initial adhesion to the silicone elastomer, it does play a major role in the growth of the basal yeast cell polylayer, vertical extension of hyphae and matrix deposition in the upper portion of the biofilm, final biofilm thickness, penetrability of human white blood cells, and final biofilm integrity (i.e., resistance to fluid flow). These results provide a more detailed description of normal biofilm development and architecture and confirm the central role played by the transcription factor Tec1 in the biofilm model employed here.

Generating a battery of monoclonal antibodies against native green fluorescent protein for immunostaining, FACS, IP, and ChIP using a unique adjuvant.

Using a unique, nontoxic adjuvant compound of poly(I:C) and anti-CD40 MAb, a battery of eight mouse monoclonal antibodies was generated against native green fluorescent protein. All were effective to varying degrees for immunostaining paraformaldehyde-fixed cells, six for staining sections of paraffin-embedded tissue, all to varying degrees in fluorescent-activated cell sorting, five for immunoprecipitation, and seven for chromatin immunoprecipitation. None worked in denaturing Western blots since the target was the native GFP protein. Both the hybridomas and antibodies are available at cost through DSHB, a non-profit National Resource created by the National Institutes of Health.

Hsp104 overexpression cures Saccharomyces cerevisiae [PSI+] by causing dissolution of the prion seeds.

The [PSI(+)] yeast prion is formed when Sup35 misfolds into amyloid aggregates. [PSI(+)], like other yeast prions, is dependent on the molecular chaperone Hsp104, which severs the prion seeds so that they pass on as the yeast cells divide. Surprisingly, however, overexpression of Hsp104 also cures [PSI(+)]. Several models have been proposed to explain this effect: inhibition of severing, asymmetric segregation of the seeds between mother and daughter cells, and dissolution of the prion seeds. First, we found that neither the kinetics of curing nor the heterogeneity in the distribution of the green fluorescent protein (GFP)-labeled Sup35 foci in partially cured yeast cells is compatible with Hsp104 overexpression curing [PSI(+)] by inhibiting severing. Second, we ruled out the asymmetric segregation model by showing that the extent of curing was essentially the same in mother and daughter cells and that the fluorescent foci did not distribute asymmetrically, but rather, there was marked loss of foci in both mother and daughter cells. These results suggest that Hsp104 overexpression cures [PSI(+)] by dissolution of the prion seeds in a two-step process. First, trimming of the prion seeds by Hsp104 reduces their size, and second, their amyloid core is eliminated, most likely by proteolysis.

Impact of environmental conditions on the form and function of Candida albicans biofilms.

Candida albicans, like other pathogens, can form complex biofilms on a variety of substrates. However, as the number of studies of gene regulation, architecture, and pathogenic traits of C. albicans biofilms has increased, so have differences in results. This suggests that depending upon the conditions employed, biofilms may vary widely, thus hampering attempts at a uniform description. Gene expression studies suggest that this may be the case. To explore this hypothesis further, we compared the architectures and traits of biofilms formed in RPMI 1640 and Spider media at 37°C in air. Biofilms formed by a/α cells in the two media differed to various degrees in cellular architecture, matrix deposition, penetrability by leukocytes, fluconazole susceptibility, and the facilitation of mating. Similar comparisons of a/a cells in the two media, however, were made difficult given that in air, although a/a cells form traditional biofilms in RPMI medium, they form polylayers composed primarily of yeast cells in Spider medium. These polylayers lack an upper hyphal/matrix region, are readily penetrated by leukocytes, are highly fluconazole susceptible, and do not facilitate mating. If, however, air is replaced with 20% CO2, a/a cells make a biofilm in Spider medium similar architecturally to that of a/α cells, which facilitates mating. A second, more cursory comparison is made between the disparate cellular architectures of a/a biofilms formed in air in RPMI and Lee's media. The results demonstrate that C. albicans forms very different types of biofilms depending upon the composition of the medium, level of CO2 in the atmosphere, and configuration of the MTL locus.

Candida albicans forms a specialized "sexual" as well as "pathogenic" biofilm.

Candida albicans forms two types of biofilm in RPMI 1640 medium, depending upon the configuration of the mating type locus. In the prevalent a/α configuration, cells form a biofilm that is impermeable, impenetrable by leukocytes, and fluconazole resistant. It is regulated by the Ras1/cyclic AMP (cAMP) pathway. In the a/a or α/α configuration, white cells form a biofilm that is architecturally similar to an a/α biofilm but, in contrast, is permeable, penetrable, and fluconazole susceptible. It is regulated by the mitogen-activated protein (MAP) kinase pathway. The MTL-homozygous biofilm has been shown to facilitate chemotropism, a step in the mating process. This has led to the hypothesis that specialized MTL-homozygous biofilms facilitate mating. If true, then MTL-homozygous biofilms should have an advantage over MTL-heterozygous biofilms in supporting mating. We have tested this prediction using a complementation strategy and show that minority opaque a/a and α/α cells seeded in MTL-homozygous biofilms mate at frequencies 1 to 2 orders of magnitude higher than in MTL-heterozygous biofilms. No difference in mating frequencies was observed between seeded patches of MTL-heterozygous and MTL-homozygous cells grown on agar at 28°C in air or 20% CO2 and at 37°C. Mating frequencies are negligible in seeded patches of both a/α and a/a cells, in contrast to seeded biofilms. Together, these results support the hypothesis that MTL-homozygous (a/a or α/α) white cells form a specialized "sexual biofilm."

Huntingtin fragments and SOD1 mutants form soluble oligomers in the cell.

Diffusion coefficients of huntingtin (Htt) fragments and SOD1 mutants expressed in cells were measured using fluorescence correlation spectroscopy. The diffusion mobilities of both non-pathological Htt fragments (25 polyQs) and pathological Htt fragments (103 polyQs) were much slower than expected for monomers suggesting that they oligomerize. The mobility of these fragments was unaffected by duration of expression or by over-expression of Hsp70 and Hsp40. However in cells with HttQ103 inclusions, diffusion measurements showed that the residual cytosolic HttQ103 was monomeric. These results suggest that both non-pathological and pathological Htt fragments form soluble oligomers in the cytosol with the properties of the oligomers determining whether they cause pathology. SOD1 with point mutations (A4V, G37R, and G85R) also had slower diffusional mobility than the wild-type protein whose mobility was consistent with that of a dimer. However, the decrease in mobility of the different SOD1 mutants did not correlate with their known pathology. Therefore, while soluble oligomers always seem to be present under conditions where cell pathology occurs, the presence of the oligomers, in itself, does not determine the extent of neuropathology.

Differences in the curing of [PSI+] prion by various methods of Hsp104 inactivation.

[PSI(+)] yeast, containing the misfolded amyloid conformation of Sup35 prion, is cured by inactivation of Hsp104. There has been controversy as to whether inactivation of Hsp104 by guanidine treatment or by overexpression of the dominant negative Hsp104 mutant, Hsp104-2KT, cures [PSI(+)] by the same mechanism- inhibition of the severing of the prion seeds. Using live cell imaging of Sup35-GFP, overexpression of Hsp104-2KT caused the foci to increase in size, then decrease in number, and finally disappear when the cells were cured, similar to that observed in cells cured by depletion of Hsp104. In contrast, guanidine initially caused an increase in foci size but then the foci disappeared before the cells were cured. By starving the yeast to make the foci visible in cells grown with guanidine, the number of cells with foci was found to correlate exactly with the number of [PSI(+)] cells, regardless of the curing method. Therefore, the fluorescent foci are the prion seeds required for maintenance of [PSI(+)] and inactivation of Hsp104 cures [PSI(+)] by preventing severing of the prion seeds. During curing with guanidine, the reduction in seed size is an Hsp104-dependent effect that cannot be explained by limited severing of the seeds. Instead, in the presence of guanidine, Hsp104 retains an activity that trims or reduces the size of the prion seeds by releasing Sup35 molecules that are unable to form new prion seeds. This Hsp104 activity may also occur in propagating yeast.

Sequestration of Sup35 by aggregates of huntingtin fragments causes toxicity of [PSI+] yeast.

Expression of huntingtin fragments with 103 glutamines (HttQ103) is toxic in yeast containing either the [PIN(+)] prion, which is the amyloid form of Rnq1, or [PSI(+)] prion, which is the amyloid form of Sup35. We find that HttQP103, which has a polyproline region at the C-terminal end of the polyQ repeat region, is significantly more toxic in [PSI(+)] yeast than in [PIN(+)], even though HttQP103 formed multiple aggregates in both [PSI(+)] and [PIN(+)] yeast. This toxicity was only observed in the strong [PSI(+)] variant, not the weak [PSI(+)] variant, which has more soluble Sup35 present than the strong variant. Furthermore, expression of the MC domains of Sup35, which retains the C-terminal domain of Sup35, but lacks the N-terminal prion domain, almost completely rescued HttQP103 toxicity, but was less effective in rescuing HttQ103 toxicity. Therefore, the toxicity of HttQP103 in yeast containing the [PSI(+)] prion is primarily due to sequestration of the essential protein, Sup35.

Application of the FLP/FRT system for conditional gene deletion in yeast Saccharomyces cerevisiae.

The yeast Saccharomyces cerevisiae has proved to be an excellent model organism to study the function of proteins. One of the many advantages of yeast is the many genetic tools available to manipulate gene expression, but there are still limitations. To complement the many methods used to control gene expression in yeast, we have established a conditional gene deletion system by using the FLP/FRT system on yeast vectors to conditionally delete specific yeast genes. Expression of Flp recombinase, which is under the control of the GAL1 promoter, was induced by galactose, which in turn excised FRT sites flanked genes. The efficacy of this system was examined using the FRT site-flanked genes HSP104, URA3 and GFP. The pre-excision frequency of this system, which might be caused by the basal activity of the GAL1 promoter or by spontaneous recombination between FRT sites, was detected ca. 2% under the non-selecting condition. After inducing expression of Flp recombinase, the deletion efficiency achieved ca. 96% of cells in a population within 9 h. After conditional deletion of the specific gene, protein degradation and cell division then diluted out protein that was expressed from this gene prior to its excision. Most importantly, the specific protein to be deleted could be expressed under its own promoter, so that endogenous levels of protein expression were maintained prior to excision by the Flp recombinase. Therefore, this system provides a useful tool for the conditional deletion of genes in yeast.

Application of GFP-labeling to study prions in yeast.

Fluorescent live cell imaging has recently been used in numerous studies to examine prions in yeast. These fluorescence studies take advantage of the fact that unlike the normally folded form, the misfolded amyloid form of the prion protein is aggregated. The studies have used fluorescence to identify new prions, to study the transmission of prion from mother to daughter, and to understand the role of molecular chaperones in this transmission. The use of fluorescence imaging complements the more standard methods used to study prion propagation. This review discusses the various studies that have taken advantage of fluorescence imaging technique particularly in regard to understanding the transmission and curing of the [PSI(+)], the prion form of the translation termination factor Sup35p.