Deenadayalan Bakthavatsalam

The Dictyostelium genome encodes numerous RasGEFs with multiple biological roles

BackgroundDictyostelium discoideum is a eukaryote with a simple lifestyle and a relatively small genome whose sequence has been fully determined. It is widely used for studies on cell signaling, movement and multicellular development. Ras guanine-nucleotide exchange factors (RasGEFs) are the proteins that activate Ras and thus lie near the top of many signaling pathways. They are particularly important for signaling in development and chemotaxis in many organisms, including Dictyostelium.ResultsWe have searched the genome for sequences encoding RasGEFs. Despite its relative simplicity, we find that the Dictyostelium genome encodes at least 25 RasGEFs, with a few other genes encoding only parts of the RasGEF consensus domains. All appear to be expressed at some point in development. The 25 genes include a wide variety of domain structures, most of which have not been seen in other organisms. The LisH domain, which is associated with microtubule binding, is seen particularly frequently; other domains that confer interactions with the cytoskeleton are also common. Disruption of a sample of the novel genes reveals that many have clear phenotypes, including altered morphology and defects in chemotaxis, slug phototaxis and thermotaxis.ConclusionThese results suggest that the unexpectedly large number of RasGEF genes reflects an evolutionary expansion of the range of Ras signaling rather than functional redundancy or the presence of multiple pseudogenes.

The secreted Dictyostelium protein CfaD is a chalone

Dictyostelium discoideum cells secrete CfaD, a protein that is similar to cathepsin proteases. Cells that lack cfaD proliferate faster and reach a higher stationary-phase density than wild-type cells, whereas cells that overexpress CfaD proliferate slowly and reach the stationary phase when at a low density. On a per-nucleus basis, CfaD affects proliferation but not growth. The drawback of not having CfaD is a reduced spore viability. Recombinant CfaD has no detectable protease activity but, when added to cells, inhibits the proliferation of wild-type and cfaD– cells. The secreted protein AprA also inhibits proliferation. AprA is necessary for the effect of CfaD on proliferation. Molecular-sieve chromatography indicates that in conditioned growth medium, the 60 kDa CfaD is part of a ∼150 kDa complex, and both chromatography and pull-down assays suggest that CfaD interacts with AprA. These results suggest that two interacting proteins may function together as a chalone signal in a negative feedback loop that slows Dictyostelium cell proliferation.

The secreted proteome profile of developing Dictyostelium discoideum cells

Dictyostelium discoideum is a unicellular eukaryote that, when starved, aggregates to form multicellular structures. In this report, we identified the proteins secreted by developing Dictyostelium cells using MS‐based proteomics. A total of 349 different secreted proteins were identified, indicating that at least 2.6% of the 13 600 predicted proteins in the Dictyostelium genome are secreted. Gene ontology analysis suggests that many of the secreted proteins are involved in protein and carbohydrate metabolism, and proteolysis.

A G protein-coupled receptor with a lipid kinase domain is involved in cell-density sensing.

One mechanism multicellular structures use for controlling cell number [1, 2] involves the secretion and sensing of a factor, such as leptin [3] or myostatin [4], in mammals. Dictyostelium cells secrete autocrine factors for sensing cell density prior to aggregation and multicellular development [5, 6] such as CMF (conditioned-medium factor), which enables starving cells to respond to cAMP pulses [7-9]. Its actions are mediated by two receptors. CMFR1 activates a G protein-independent signaling pathway regulating gene expression [10]. An unknown Galpha1-dependent receptor activates phospholipase C (PLC), which regulates the lifetime of Galpha2-GTP [11-13]. Here, we describe RpkA, an unusual seven-transmembrane receptor that is fused to a C-terminal PIP5 kinase domain and that localizes in membranes of a late endosomal compartment. Loss of RpkA resulted in formation of persistent loose aggregates and altered expression of cAMP-regulated genes. The developmental defect can be rescued by full-length RpkA and the transmembrane domain only. The PIP5 kinase domain is dispensable for the developmental role of RpkA. rpkA- cells secrete and bind CMF but are unable to induce downstream responses. Inactivation of Galpha1, a negative regulator of CMF signaling, rescued the developmental defect of the rpkA- cells, suggesting that RpkA actions are mediated by Galpha1.

A Dictyostelium chalone uses G proteins to regulate proliferation

BackgroundSeveral studies have shown that organ size, and the proliferation of tumor metastases, may be regulated by negative feedback loops in which autocrine secreted factors called chalones inhibit proliferation. However, very little is known about chalones, and how cells sense them. We previously identified two secreted proteins, AprA and CfaD, which act as chalones in Dictyostelium. Cells lacking AprA or CfaD proliferate faster than wild-type cells, and adding recombinant AprA or CfaD to cells slows their proliferation.ResultsWe show here that cells lacking the G protein components Galpha8, Galpha9, and Gbeta proliferate faster than wild-type cells despite secreting normal or high levels of AprA and CfaD. Compared with wild-type cells, the proliferation of galpha8-, galpha9- and gbeta- cells are only weakly inhibited by recombinant AprA (rAprA). Like AprA and CfaD, Galpha8 and Gbeta inhibit cell proliferation but not cell growth (the rate of increase in mass and protein per nucleus), whereas Galpha9 inhibits both proliferation and growth. galpha8- cells show normal cell-surface binding of rAprA, whereas galpha9- and gbeta- cells have fewer cell-surface rAprA binding sites, suggesting that Galpha9 and Gbeta regulate the synthesis or processing of the AprA receptor. Like other ligands that activate G proteins, rAprA induces the binding of [3H]GTP to membranes, and GTPgammaS inhibits the binding of rAprA to membranes. Both AprA-induced [3H]GTP binding and the GTPgammaS inhibition of rAprA binding require Galpha8 and Gbeta but not Galpha9. Like aprA- cells, galpha8- cells have reduced spore viability.ConclusionThis study shows that Galpha8 and Gbeta are part of the signal transduction pathway used by AprA to inhibit proliferation but not growth in Dictyostelium, whereas Galpha9 is part of a differealnt pathway that regulates both proliferation and growth, and that a chalone signal transduction pathway uses G proteins.

Dictyostelium cells bind a secreted autocrine factor that represses cell proliferation

BackgroundDictyostelium cells secrete the proteins AprA and CfaD. Cells lacking either AprA or CfaD proliferate faster than wild type, while AprA or CfaD overexpressor cells proliferate slowly, indicating that AprA and CfaD are autocrine factors that repress proliferation. CfaD interacts with AprA and requires the presence of AprA to slow proliferation. To determine if CfaD is necessary for the ability of AprA to slow proliferation, whether AprA binds to cells, and if so whether the binding requires the presence of CfaD, we examined the binding and effect on proliferation of recombinant AprA.ResultsWe find that the extracellular accumulation of AprA increases with cell density and reaches a concentration of 0.3 μg/ml near a stationary cell density. When added to wild-type or aprA- cells, recombinant AprA (rAprA) significantly slows proliferation at 0.1 μg/ml and higher concentrations. From 4 to 64 μg/ml, the effect of rAprA is at a plateau, slowing but not stopping proliferation. The proliferation-inhibiting activity of rAprA is roughly the same as that of native AprA in conditioned growth medium. Proliferating aprA- cells show saturable binding of rAprA to 92,000 ± 11,000 cell-surface receptors with a KDof 0.03 ± 0.02 μg/ml. There appears to be one class of binding site, and no apparent cooperativity. Native AprA inhibits the binding of rAprA to aprA- cells with a Kiof 0.03 μg/ml, suggesting that the binding kinetics of rAprA are similar to those of native AprA. The proliferation of cells lacking CrlA, a cAMP receptor-like protein, or cells lacking CfaD are not affected by rAprA. Surprisingly, both cell types still bind rAprA.ConclusionTogether, the data suggest that AprA functions as an autocrine proliferation-inhibiting factor by binding to cell surface receptors. Although AprA requires CfaD for activity, it does not require CfaD to bind to cells, suggesting the possibility that cells have an AprA receptor and a CfaD receptor, and activation of both receptors is required to slow proliferation. We previously found that crlA- cells are sensitive to CfaD. Combined with the results presented here, this suggests that CrlA is not the AprA or CfaD receptor, and may be the receptor for an unknown third factor that is required for AprA and CfaD activity.

Linking Ras to myosin function: RasGEF Q, a Dictyostelium exchange factor for RasB, affects myosin II functions

Ras guanine nucleotide exchange factor (GEF) Q, a nucleotide exchange factor from Dictyostelium discoideum, is a 143-kD protein containing RasGEF domains and a DEP domain. We show that RasGEF Q can bind to F-actin, has the potential to form complexes with myosin heavy chain kinase (MHCK) A that contain active RasB, and is the predominant exchange factor for RasB. Overexpression of the RasGEF Q GEF domain activates RasB, causes enhanced recruitment of MHCK A to the cortex, and leads to cytokinesis defects in suspension, phenocopying cells expressing constitutively active RasB, and myosin-null mutants. RasGEF Q− mutants have defects in cell sorting and slug migration during later stages of development, in addition to cell polarity defects. Furthermore, RasGEF Q− mutants have increased levels of unphosphorylated myosin II, resulting in myosin II overassembly. Collectively, our results suggest that starvation signals through RasGEF Q to activate RasB, which then regulates processes requiring myosin II.

A Cell Number-Counting Factor Regulates Levels of a Novel Protein, SslA, as Part of a Group Size Regulation Mechanism in Dictyostelium

ABSTRACT Developing Dictyostelium cells form aggregation streams that break into groups of ∼2 × 104 cells. The breakup and subsequent group size are regulated by a secreted multisubunit counting factor (CF). To elucidate how CF regulates group size, we isolated second-site suppressors of smlA−, a transformant that forms small groups due to oversecretion of CF. smlA−sslA1(CR11) cells form roughly wild-type-size groups due to an insertion in the beginning of the coding region of sslA1, one of two highly similar genes encoding a novel protein. The insertion increases levels of SslA. In wild-type cells, the sslA1(CR11) mutation forms abnormally large groups. Reducing SslA levels by antisense causes the formation of smaller groups. The sslA(CR11) mutation does not affect the extracellular accumulation of CF activity or the CF components countin and CF50, suggesting that SslA does not regulate CF secretion. However, CF represses levels of SslA. Wild-type cells starved in the presence of smlA− cells, recombinant countin, or recombinant CF50 form smaller groups, whereas sslA1(CR11) cells appear to be insensitive to the presence of smlA− cells, countin, or CF50, suggesting that the sslA1(CR11) insertion affects CF signal transduction. We previously found that CF reduces intracellular glucose levels. sslA(CR11) does not significantly affect glucose levels, while glucose increases SslA levels. Together, the data suggest that SslA is a novel protein involved in part of a signal transduction pathway regulating group size.

A retinoblastoma orthologue is required for the sensing of a chalone in Dictyostelium discoideum.

ABSTRACT Retinoblastoma-like proteins regulate cell differentiation and inhibit cell proliferation. The Dictyostelium discoideum retinoblastoma orthologue RblA affects the differentiation of cells during multicellular development, but it is unclear whether RblA has a significant effect on Dictyostelium cell proliferation, which is inhibited by the secreted proteins AprA and CfaD. We found that rblA− cells in shaking culture proliferate to a higher density, die faster after reaching stationary density, and, after starvation, have a lower spore viability than wild-type cells, possibly because in shaking culture, rblA− cells have both increased cytokinesis and lower extracellular accumulation of CfaD. However, rblA− cells have abnormally slow proliferation on bacterial lawns. Recombinant AprA inhibits the proliferation of wild-type cells but not that of rblA− cells, whereas CfaD inhibits the proliferation of both wild-type cells and rblA− cells. Similar to aprA− cells, rblA− cells have a normal mass and protein accumulation rate on a per-nucleus basis, indicating that RblA affects cell proliferation but not cell growth. AprA also functions as a chemorepellent, and RblA is required for proper AprA chemorepellent activity despite the fact that RblA does not affect cell speed. Together, our data indicate that an autocrine proliferation-inhibiting factor acts through RblA to regulate cell density in Dictyostelium, suggesting that such factors may signal through retinoblastoma-like proteins to control the sizes of structures such as developing organs or tumors.