Dictyostelium Aggregation Research Articles

Loss of mfsd8 alters the secretome during Dictyostelium aggregation

Major facilitator superfamily domain-containing protein 8 (MFSD8) is a transmembrane protein that has been reported to function as a lysosomal chloride channel. In humans, homozygous mutations in MFSD8 cause a late-infantile form of neuronal ceroid lipofuscinosis (NCL) called CLN7 disease. In the social amoeba Dictyostelium discoideum, Mfsd8 localizes to cytoplasmic puncta and vesicles, and regulates conserved processes during the organism’s life cycle. Here, we used D. discoideum to examine the effect of mfsd8-deficiency on the secretome during the early stages of multicellular development. Mass spectrometry revealed 61 proteins that were differentially released by cells after 4 and 8 h of starvation. Most proteins were present in increased amounts in mfsd8- conditioned buffer compared to WT indicating that loss of mfsd8 deregulates protein secretion and/or causes the release of proteins not normally secreted by WT cells. GO term enrichment analyses showed that many of the proteins aberrantly released by mfsd8- cells localize to compartments and regions of the cell associated with the endo-lysosomal and secretory pathways. Mass spectrometry also revealed proteins previously known to be impacted by the loss of mfsd8 (e.g., cathepsin D), as well as proteins that may underlie mfsd8-deficiency phenotypes during aggregation. Finally, we show that mfsd8-deficiency reduces intracellular proteasome 20S activity due to the abnormal release of at least one proteasomal subunit. Together, this study reveals the impact of mfsd8 loss on the secretome during D. discoideum aggregation and lays the foundation for follow up work that investigates the role of altered protein release in CLN7 disease.

Development of a Positive Selection High Throughput Genetic Screen in Dictyostelium discoideum.

The cellular slime mold Dictyostelium discoideum is a powerful model organism that can be utilized to investigate human health and disease. One particular strength of Dictyostelium is that it can be utilized for high throughput genetic screens. For many phenotypes, one limitation of utilizing Dictyostelium is that screening can be an arduous and time-consuming process, limiting the genomic depth one can cover. Previously, we utilized a restriction enzyme-mediated integration screen to identify suppressors of polyglutamine aggregation in Dictyostelium. However, due to the time required to perform the screen, we only obtained ∼4% genome coverage. Here we have developed an efficient screening pipeline that couples chemical mutagenesis with the 5-fluoroorotic acid counterselection system to enrich for mutations in genes of interest. Here we describe this new screening methodology and highlight how it can be utilized for other biological systems.

Simulation modeling of cAMP induced Dictyostelium aggregation by using object-oriented Pharo programming language

Simulation modeling of cAMP induced Dictyostelium aggregation by using object-oriented Pharo programming language

DPF is a cell-density sensing factor, with cell-autonomous and non-autonomous functions during Dictyostelium growth and development

BackgroundCellular functions can be regulated by cell-cell interactions that are influenced by extra-cellular, density-dependent signaling factors. Dictyostelium grow as individual cells in nutrient-rich sources, but, as nutrients become depleted, they initiate a multi-cell developmental program that is dependent upon a cell-density threshold. We hypothesized that novel secreted proteins may serve as density-sensing factors to promote multi-cell developmental fate decisions at a specific cell-density threshold, and use Dictyostelium in the identification of such a factor.ResultsWe show that multi-cell developmental aggregation in Dictyostelium is lost upon minimal (2-fold) reduction in local cell density. Remarkably, developmental aggregation response at non-permissive cell densities is rescued by addition of conditioned media from high-density, developmentally competent cells. Using rescued aggregation of low-density cells as an assay, we purified a single, 150-kDa extra-cellular protein with density aggregation activity. MS/MS peptide sequence analysis identified the gene sequence, and cells that overexpress the full-length protein accumulate higher levels of a development promoting factor (DPF) activity than parental cells, allowing cells to aggregate at lower cell densities; cells deficient for this DPF gene lack density-dependent developmental aggregation activity and require higher cell density for cell aggregation compared to WT. Density aggregation activity co-purifies with tagged versions of DPF and tag-affinity-purified DPF possesses density aggregation activity. In mixed development with WT, cells that overexpress DPF preferentially localize at centers for multi-cell aggregation and define cell-fate choice during cytodifferentiation. Finally, we show that DPF is synthesized as a larger precursor, single-pass transmembrane protein, with the p150 fragment released by proteolytic cleavage and ectodomain shedding. The TM/cytoplasmic domain of DPF possesses cell-autonomous activity for cell-substratum adhesion and for cellular growth.ConclusionsWe have purified a novel secreted protein, DPF, that acts as a density-sensing factor for development and functions to define local collective thresholds for Dictyostelium development and to facilitate cell-cell communication and multi-cell formation. Regions of high DPF expression are enriched at centers for cell-cell signal-response, multi-cell formation, and cell-fate determination. Additionally, DPF has separate cell-autonomous functions for regulation of cellular adhesion and growth.

A telomerase with novel non-canonical roles: TERT controls cellular aggregation and tissue size in Dictyostelium.

Telomerase, particularly its main subunit, the reverse transcriptase, TERT, prevents DNA erosion during eukaryotic chromosomal replication, but also has poorly understood non-canonical functions. Here, in the model social amoeba Dictyostelium discoideum, we show that the protein encoded by tert has telomerase-like motifs, and regulates, non-canonically, important developmental processes. Expression levels of wild-type (WT) tert were biphasic, peaking at 8 and 12 h post-starvation, aligning with developmental events, such as the initiation of streaming (~7 h) and mound formation (~10 h). In tert KO mutants, however, aggregation was delayed until 16 h. Large, irregular streams formed, then broke up, forming small mounds. The mound-size defect was not induced when a KO mutant of countin (a master size-regulating gene) was treated with TERT inhibitors, but anti-countin antibodies did rescue size in the tert KO. Although, conditioned medium (CM) from countin mutants failed to rescue size in the tert KO, tert KO CM rescued the countin KO phenotype. These and additional observations indicate that TERT acts upstream of smlA/countin: (i) the observed expression levels of smlA and countin, being respectively lower and higher (than WT) in the tert KO; (ii) the levels of known size-regulation intermediates, glucose (low) and adenosine (high), in the tert mutant, and the size defect’s rescue by supplemented glucose or the adenosine-antagonist, caffeine; (iii) the induction of the size defect in the WT by tert KO CM and TERT inhibitors. The tert KO’s other defects (delayed aggregation, irregular streaming) were associated with changes to cAMP-regulated processes (e.g. chemotaxis, cAMP pulsing) and their regulatory factors (e.g. cAMP; acaA, carA expression). Overexpression of WT tert in the tert KO rescued these defects (and size), and restored a single cAMP signaling centre. Our results indicate that TERT acts in novel, non-canonical and upstream ways, regulating key developmental events in Dictyostelium.

Synthetic Chemotaxis and Collective Behavior in Active Matter.

The ability to navigate in chemical gradients, called chemotaxis, is crucial for the survival of microorganisms. It allows them to find food and to escape from toxins. Many microorganisms can produce the chemicals to which they respond themselves and use chemotaxis for signaling, which can be seen as a basic form of communication, allowing ensembles of microorganisms to coordinate their behavior, for example, during embryogenesis, biofilm formation, or cellular aggregation. For example, Dictyostelium cells use signaling as a survival strategy: when starving, they produce certain chemicals toward which other cells show taxis. This leads to aggregation of the cells resulting in a multicellular aggregate that can sustain long starvation periods. Remarkably, the past decade has led to the development of synthetic microswimmers, which can self-propel through a solvent, analogously to bacteria and other microorganisms. The mechanism underlying the self-propulsion of synthetic microswimmers like camphor boats, droplet swimmers, and in particular autophoretic Janus colloids involves the production of certain chemicals. As we will discuss in this Account, the same chemicals (phoretic fields) involved in the self-propulsion of a (Janus) microswimmer also act on other ones and bias their swimming direction toward (or away from) the producing microswimmer. Synthetic microswimmers therefore provide a synthetic analogue to motile microorganisms interacting by taxis toward (or away from) self-produced chemical fields. In this Account, we review recent progress in the theoretical description of synthetic chemotaxis mainly based on simulations and field theoretical descriptions. We will begin with single motile particles leaving chemical trails behind with which they interact themselves, leading to effects like self-trapping or self-avoidance. Besides these self-interactions, in ensembles of synthetic motile particles each particle also responds to the chemicals produced by other particles, inducing chemical (or phoretic) cross-interactions. When these interactions are attractive, they commonly lead to clusters, even at low particle density. These clusters may either proceed toward macrophase separation, resembling Dictyostelium aggregation, or, as shown very recently, lead to dynamic clusters of self-limited size (dynamic clustering) as seen in experiments in autophoretic Janus colloids. Besides the classical case where chemical interactions are attractive, this Account discusses, as its main focus, repulsive chemical interactions, which can create a new and less known avenue to pattern formation in active systems leading to a variety of pattern, including clusters which are surrounded by shells of chemicals, traveling waves and more complex continuously reshaping patterns. In all these cases "synthetic signalling" can crucially determine the collective behavior of synthetic microswimmer ensembles and can be used as a design principle to create patterns in motile active particles.

Effects of deletion of the receptor CrlA on Dictyostelium aggregation and MPBD-mediated responses are strain dependent and not evident in strain Ax2

The polyketide MPBD (4-methyl-5-pentylbenzene-1, 3-diol) is produced by the polyketide synthase SteelyA (StlA) in Dictyostelium discoideum. MPBD is required for appropriate expression of cAMP signalling genes involved in cell aggregation and additionally induces the spore maturation at the fruiting body stage. The MPBD signalling pathway for regulation of cell aggregation is unknown, but MPBD effects on sporulation were reported to be mediated by the G-protein coupled receptor CrlA in D. discoideum KAx3. In this study, we deleted the crlA gene from the same parental strain (Ax2) that was used to generate the MPBD-less mutant. We found that unlike the MPBD-less mutant, Ax2-derived crlA- mutants exhibited normal cell aggregation, indicating that in Ax2 MPBD effects on early development do not require CrlA. We also found that the Ax2/crlA- mutant formed normal spores in fruiting bodies. When transformed with PkaC, both Ax2 and Ax2/crlA- similarly responded to MPBD in vitro with spore encapsulation. Our data make it doubtful that CrlA acts as the receptor for MPBD signalling during the development of D. discoideum Ax2.

Directional sensing and streaming in Dictyostelium aggregation.

We merge the Kessler-Levine simple discrete model for Dictyostelium cyclic adenosine monophosphate (cAMP) production and diffusion with the Dilão-Hauser directional sensing aggregation mechanism. The resulting compound model describes all the known transient patterns that emerge during Dictyostelium aggregation, which include the spontaneous formation of cAMP self-sustained target and spiral waves and streaming. We show that the streaming patterns depend on the speed of the amoebae, on the relaxation time for the production of cAMP, on the cAMP degradation rate, and on directional sensing. Moreover, we show that different signaling centers emerge during Dictyostelium aggregation.

A High-Throughput, Multi-Cell Phenotype Assay for the Identification of Novel Inhibitors of Chemotaxis/Migration

Chemotaxis and cell migration are fundamental, universal eukaryotic processes essential for biological functions such as embryogenesis, immunity, cell renewal, and wound healing, as well as for pathogenesis of many diseases including cancer metastasis and chronic inflammation. To identify novel chemotaxis inhibitors as probes for mechanistic studies and leads for development of new therapeutics, we developed a unique, unbiased phenotypic chemotaxis-dependent Dictyostelium aggregation assay for high-throughput screening using rapid, laser-scanning cytometry. Under defined conditions, individual Dictyostelium secrete chemoattractants, migrate, and aggregate. Chemotaxis is quantified by laser-scanning cytometry with a GFP marker expressed only in cells after chemotaxis/multi-cell aggregation. We applied the assay to screen 1,280 known compounds in a 1536-well plate format and identified two chemotaxis inhibitors. The chemotaxis inhibitory activities of both compounds were confirmed in both Dictyostelium and in human neutrophils in a directed EZ-TAXIscan chemotaxis assay. The compounds were also shown to inhibit migration of two human cancer cell lines in monolayer scratch assays. This test screen demonstrated that the miniaturized assay is extremely suited for high-throughput screening of very large libraries of small molecules to identify novel classes of chemotaxis/migratory inhibitors for drug development and research tools for targeting chemotactic pathways universal to humans and other systems.

Chemotaxis with directional sensing during Dictyostelium aggregation

We show that the chemotactic movements of colonies of the starving amoeba Dictyostelium discoideum are driven by a force that depends on both the direction of propagation (directional sensing) of reaction-diffusion chemotactic waves and on the gradient of the concentration of the chemoattractant, solving the chemotactic wave paradox. It is shown that the directional sensing of amoebae is due to the sensitivity of the cells to the time variation of the concentration of the chemoattractant combined with its spatial gradient. It is also shown that chemotaxis exclusively driven by local concentration gradient leads to unstable local motion, preventing cells from aggregation. These findings show that the formation of mounds, which initiate multicellularity in Dictyostelium discoideum, is caused by the sensitivity of the amoebae due to three factors, namely, to the direction of propagation of the chemoattractant, to its spatial gradient, and to the emergence of cAMP “emitting centres”, responsible for the local accumulation of the amoebae.

Determinants of RasC Specificity during Dictyostelium Aggregation

RasC is required for optimum activation of adenylyl cyclase A and for aggregate stream formation during the early differentiation of Dictyostelium discoideum. RasG is unable to substitute for this requirement despite its sequence similarity to RasC. A critical question is which amino acids in RasC are required for its specific function. Each of the amino acids within the switch 1 and 2 domains in the N-terminal portion of RasG was changed to the corresponding amino acid from RasC, and the ability of the mutated RasG protein to reverse the phenotype of rasC(-) cells was determined. Only the change from aspartate at position 30 of RasG to alanine (the equivalent position 31 in RasC) resulted in a significant increase in adenylyl cyclase A activation and a partial reversal of the aggregation-deficient phenotype of rasC(-) cells. All other single amino acid changes were without effect. Expression of a chimeric protein, RasG(1-77)-RasC(79-189), also resulted in a partial reversal of the rasC(-) cell phenotype, indicating the importance of the C-terminal portion of RasC. Furthermore, expression of the chimeric protein, with alanine changed to aspartate (RasG(1-77(D30A))-RasC(79-189)), resulted in a full rescue the rasC(-) aggregation-deficient phenotype. Finally, the expression of either a mutated RasC, with the aspartate 31 replaced by alanine, or the chimeric protein, RasC(1-78)-RasG(78-189), only generated a partial rescue. These results emphasize the importance of both the single amino acid at position 31 and the C-terminal sequence for the specific function of RasC during Dictyostelium aggregation.

Computational modelling suggests dynamic interactions between Ca2+, IP3 and G protein-coupled modules are key to robust Dictyostelium aggregation

Under conditions of starvation, Dictyostelium cells begin a programme of development during which they aggregate to form a multicellular structure by chemotaxis, guided by propagating waves of cyclic AMP that are relayed robustly from cell to cell. In this paper, we develop and analyse a new model for the intracellular and extracellular cAMP dependent processes that regulate Dictyostelium migration. The model allows, for the first time, a quantitative analysis of the dynamic interactions between calcium, IP(3) and G protein-dependent modules that are shown to be key to the generation of robust cAMP oscillations in Dictyostelium cells. The model provides a mechanistic explanation for the transient increase in cytosolic free Ca(2+) concentration seen in recent experiments with the application of the calmodulin inhibitor calmidazolium (R24571) to Dictyostelium cells, and also allows elucidation of the effects of varying both the conductivity of stretch-activated channels and the concentration of external phosphodiesterase on the oscillatory regime of an individual cell. A rigorous analysis of the robustness of the new model shows that interactions between the different modules significantly reduce the sensitivity of the resulting cAMP oscillations to variations in the kinetics of different Dictyostelium cells, an essential requirement for the generation of the spatially and temporally synchronised chemoattractant cAMP waves that guide Dictyostelium aggregation.

Rap1 Activation in Response to cAMP Occurs Downstream of Ras Activation during Dictyostelium Aggregation

We have used a doubly disrupted rasC(-)/rasG(-) strain of Dictyostelium discoideum, which ectopically expresses the carA gene, to explore the relationship between the activation of RasC and RasG, the two proteins that are necessary for optimum cAMP signaling, and the activation of Rap1, a Ras subfamily protein, that is also activated by cAMP. The ectopic expression of carA restored early developmental gene expression to the rasC(-)/rasG(-) strain, rendering it suitable for an analysis of cAMP signal transduction. Because there was negligible signaling through both the cAMP chemotactic pathway and the adenylyl cyclase activation pathway in the rasC(-)/rasG(-)/[act15]:carA strain, it is clear that RasG and RasC are the only two Ras subfamily proteins that directly control these pathways. The position of Rap1 in the signal transduction cascade was clarified by the finding that Rap1 activation was totally abolished in rasC(-)/rasG(-)/[act15]:carA and rasG(-) cells but only slightly reduced in rasC(-) cells. Rap1 activation, therefore, occurs downstream of the Ras proteins and predominantly, if not exclusively, downstream of RasG. The finding that in vitro guanylyl cyclase activation is also abolished in the rasC(-)/rasG(-)/[act15]:carA strain identifies RasG/RasC as the presumptive monomeric GTPases required for this activation.

Stochastic Noise and Synchronisation during Dictyostelium Aggregation Make cAMP Oscillations Robust

Stable and robust oscillations in the concentration of adenosine 3′, 5′-cyclic monophosphate (cAMP) are observed during the aggregation phase of starvation-induced development in Dictyostelium discoideum. In this paper we use mathematical modelling together with ideas from robust control theory to identify two factors which appear to make crucial contributions to ensuring the robustness of these oscillations. Firstly, we show that stochastic fluctuations in the molecular interactions play an important role in preserving stable oscillations in the face of variations in the kinetics of the intracellular network. Secondly, we show that synchronisation of the aggregating cells through the diffusion of extracellular cAMP is a key factor in ensuring robustness of the oscillatory waves of cAMP observed in Dictyostelium cell cultures to cell-to-cell variations. A striking and quite general implication of the results is that the robustness analysis of models of oscillating biomolecular networks (circadian clocks, Ca2+ oscillations, etc.) can only be done reliably by using stochastic simulations, even in the case where molecular concentrations are very high.

Streamless aggregation of Dictyostelium in the presence of isopropylidenadenosin

Starving cells of Dictyostelium discoideum undergo a developmental cycle were cAMP is autocatalytically produced and relayed from cell to cell, resulting in the propagation of excitation waves over a spatially extended population. Later on the homogeneous cell layer transforms into a pattern of cell streams directed perpendicular to the cAMP waves. Here we chemically influence aggregation competent cells by isopropylidenadenosin (IPA), an adenosine derivative. It can be assumed, that IPA acts via specific adenosine binding sites localized in the cellular membrane. We find, however, that pattern formation and cellular aggregation under the influence of IPA differ considerably compared to experiments with adenosine. In particular, our observations point towards an inhibitory effect on adenylate cyclase (ACA), the key enzyme in the autocatalytic production process of cAMP inside the cell. Our results suggest the existence of a direct coupling (via intracellular affection) or indirect coupling (via inhibition of cAMP binding) of the specific adenosine receptors to the regulatory circuit that controls cyclic intra- and extracellular cAMP concentration.

Abp1 regulates pseudopodium number in chemotaxing Dictyostelium cells

When starved, Dictyostelium cells respond to extracellular signals, polarize, and move with strong persistence into aggregation centers. Actin and actin-associated proteins play key roles in regulating both the morphology and directed movements of cells during chemotactic aggregation. Recently, we identified an ortholog of Abp1 in Dictyostelium (Dabp1). The first actin binding protein identified in yeast, Abp1 functions in actin-based endocytosis in yeast and in receptor-mediated endocytosis in mammalian cells. To explore the functions for Abp1 in Dictyostelium, we examined the phenotypes of cells that overexpressed the Dabp1 protein and cells that eliminated Dabp1 expression. In these mutants, most actin-based processes were intact. However, cell motility was altered during early development. During chemotactic streaming, more than 90% of wild-type cells had a single leading pseudopodium and a single uropodium, whereas more than 27% of Dabp1 null cells projected multiple pseuodpodia. Similarly, approximately 90% of cells that overexpressed Dabp1 projected multiple pseudopodia during chemotactic streaming, and displayed reduced rates of cell movement. Expression of the SH3 domain of Dabp1 showed this domain to be an important determinant in regulating pseudopodium number. These results suggest that Abp1 controls pseudopodium number and motility in early stages of chemotactic aggregation in Dictyostelium.

Dictyostelium CBP3 associates with actin cytoskeleton and is related to slug migration

Calcium-binding protein 3 (CBP3) expression was up-regulated under the control of the actin 15 promoter and down-regulated by RNA interference in Dictyostelium discoideum. The overexpression of CBP3 accelerated cell aggregation and formed small aggregates and fruiting body. CBP3-inhibited cells showed uneven aggregation and increased slug trail lengths toward the directed light, whereas CBP3-overexpressing cells showed the opposite phenomena. Under dark condition, the enhanced slug trail length was also observed in the CBP3-inhibited cells. Yeast two-hybrid screening identified actin 8 as interacting protein with CBP3. The interaction between CBP3 and actin was confirmed by β-galactosidase assay and surface plasmon resonance. CBP3 was associated with Triton X-100-insoluble cytoskeleton in the presence of Ca 2+ and the interaction of CBP3 with cytoskeleton was increased by the addition of Ca 2+. Using fluorescence microscopy, CBP3 was also shown to associate with the actin cytoskeleton during development. Subcellular fractionation indicated that CBP3 was enriched in cytosolic fraction. Taken together, these results suggest that CBP3 interacts with actin cytoskeleton and has a role during cell aggregation and slug migration of Dictyostelium.

Transition from an excitable to an oscillatory statein Dictyostelium discoideum

Under conditions of starvation, populations of the amoebae Dictyostelium discoideum aggregate are mediated by chemical excitation waves of cAMP. Two types of waves can be observed, either spiral or circular-shaped ones. We investigate transitions from rotating spirals to circular shaped waves (target patterns). Two different experiments demonstrating this phenomenon are presented. In the first case a continuous transition from the spiral type pattern to target waves was observed at the later stages of aggregation. In the second case the transition was induced by annihilation of waves by a spatially homogeneous cAMP pulse. Instead of the originally present spiral waves, oscillating spots bearing target patterns emerged. On the basis of a model for Dictyostelium aggregation, we provide a theoretical explanation for such transitions. It is shown that cell density can be an effective bifurcation parameter. Under certain conditions, the system is shifted from the excitable to the oscillatory state while the frequency of oscillations is proportional to the square root of the cell density. Thus, the regions with the highest cell density during the early stages of the spatial rearrangement of the cells become pacemakers and produce target patterns. The analytic results were confirmed in numerical simulations of the model.

Stochastic noise and synchronisation during Dictyostelium aggregation make cAMP oscillations robust

Stable and robust oscillations in the concentration of adenosine 3′, 5′-cyclic monophosphate (cAMP) are observed during the aggregation phase of starvation-induced development in Dictyostelium discoideum. In this paper we use mathematical modelling together with ideas from robust control theory to identify two factors which appear to make crucial contributions to ensuring the robustness of these oscillations. Firstly, we show that stochastic fluctuations in the molecular interactions play an important role in preserving stable oscillations in the face of variations in the kinetics of the intracellular network. Secondly, we show that synchronisation of the aggregating cells through the diffusion of extracellular cAMP is a key factor in ensuring robustness of the oscillatory waves of cAMP observed in Dictyostelium cell cultures to cell-to-cell variations. A striking and quite general implication of the results is that the robustness analysis of models of oscillating biomolecular networks (circadian clocks, Ca2+ oscillations, etc.) can only be done reliably by using stochastic simulations, even in the case where molecular concentrations are very high.

The different components of a multisubunit cell number-counting factor have both unique and overlapping functions.

Dictyostelium aggregation streams break up into groups of 10(3) to 2 x 10(4) cells. The cells sense the number of cells in a stream or group by the level of a secreted counting factor (CF). CF is a complex of at least 5 polypeptides. When the gene encoding countin (one of the CF polypeptides) was disrupted, the cells could not sense each other's presence, resulting in non-breaking streams that coalesced into abnormally large groups. To understand the function of the components of CF, we have isolated cDNA sequences encoding a second component of CF, CF50. CF50 is 30% identical to lysozyme (but has very little lysozyme activity) and contains distinctive serine-glycine motifs. Transformants with a disrupted cf50 gene, like countin(-) cells, form abnormally large groups. Addition of recombinant CF50 protein to developing cf50(-) cells rescues their phenotype by decreasing group size. Abnormalities seen in aggregating countin(-) cells (such as high cell-cell adhesion and low motility) are also observed in the cf50(-) cells. Western blot analysis of conditioned medium sieve column fractions showed that the CF50 protein is present in the same fraction as the 450 kDa CF complex. In the absence of CF50, secreted countin is degraded, suggesting that one function of CF50 may be to protect countin from degradation. However, unlike countin(-) cells, cf50(-) cells differentiate into an abnormally high percentage of cells expressing SP70 (a marker expressed in a subset of prespore cells), and this difference can be rescued by exposing cells to recombinant CF50. These observations indicate that unlike other known multisubunit factors, CF contains subunits with both overlapping and unique properties.