Michael A. Myre

Reconstitution of the mitochondrial calcium uniporter in yeast

Significance The mitochondrial uniporter is a highly selective calcium channel found in many diverse eukaryotes, but absent in the yeast Saccharomyces cerevisiae. Although the uniporter’s existence was recognized more than 50 y ago, its molecular components have been identified only recently. Here we use yeast as a facile reconstitution system to identify the minimal components sufficient for in vivo uniporter activity. We describe the simplified calcium uniporter of slime mold, consisting of one transmembrane component, DdMCU, which alone is sufficient for robust calcium uptake in yeast mitochondria. Intriguingly, the human uniporter requires two proteins, MCU and the animal-specific protein EMRE, that together are sufficient for uniporter activity. Our work provides a powerful reconstitution system for studying the evolution and function of this channel. The mitochondrial calcium uniporter is a highly selective calcium channel distributed broadly across eukaryotes but absent in the yeast Saccharomyces cerevisiae. The molecular components of the human uniporter holocomplex (uniplex) have been identified recently. The uniplex consists of three membrane-spanning subunits –mitochondrial calcium uniporter (MCU), its paralog MCUb, and essential MCU regulator (EMRE)– and two soluble regulatory components–MICU1 and its paralog MICU2. The minimal components sufficient for in vivo uniporter activity are unknown. Here we consider Dictyostelium discoideum (Dd), a member of the Amoebazoa outgroup of Metazoa and Fungi, and show that it has a highly simplified uniporter machinery. We show that D. discoideum mitochondria exhibit membrane potential-dependent calcium uptake compatible with uniporter activity, and also that expression of DdMCU complements the mitochondrial calcium uptake defect in human cells lacking MCU or EMRE. Moreover, expression of DdMCU in yeast alone is sufficient to reconstitute mitochondrial calcium uniporter activity. Having established yeast as an in vivo reconstitution system, we then reconstituted the human uniporter. We show that coexpression of MCU and EMRE is sufficient for uniporter activity, whereas expression of MCU alone is insufficient. Our work establishes yeast as a powerful in vivo reconstitution system for the uniporter. Using this system, we confirm that MCU is the pore-forming subunit, define the minimal genetic elements sufficient for metazoan and nonmetazoan uniporter activity, and provide valuable insight into the evolution of the uniporter machinery.

An evolutionary recent neuroepithelial cell adhesion function of huntingtin implicates ADAM10-Ncadherin

The Huntingtons disease gene product, huntingtin, is indispensable for neural tube formation, but its role is obscure. We studied neurulation in htt-null embryonic stem cells and htt-morpholino zebrafish embryos and found a previously unknown, evolutionarily recent function for this ancient protein. We found that htt was essential for homotypic interactions between neuroepithelial cells; it permitted neurulation and rosette formation by regulating metalloprotease ADAM10 activity and Ncadherin cleavage. This function was embedded in the N terminus of htt and was phenocopied by treatment of htt knockdown zebrafish with an ADAM10 inhibitor. Notably, in htt-null cells, reversion of the rosetteless phenotype occurred only with expression of evolutionarily recent htt heterologues from deuterostome organisms. Conversely, all of the heterologues that we tested, including htt from Drosophila melanogaster and Dictyostelium discoideum, exhibited anti-apoptotic activity. Thus, anti-apoptosis may have been one of htts ancestral function(s), but, in deuterostomes, htt evolved to acquire a unique regulatory activity for controlling neural adhesion via ADAM10-Ncadherin, with implications for brain evolution and development.

Deficiency of Huntingtin Has Pleiotropic Effects in the Social Amoeba Dictyostelium discoideum

Huntingtin is a large HEAT repeat protein first identified in humans, where a polyglutamine tract expansion near the amino terminus causes a gain-of-function mechanism that leads to selective neuronal loss in Huntingtons disease (HD). Genetic evidence in humans and knock-in mouse models suggests that this gain-of-function involves an increase or deregulation of some aspect of huntingtins normal function(s), which remains poorly understood. As huntingtin shows evolutionary conservation, a powerful approach to discovering its normal biochemical role(s) is to study the effects caused by its deficiency in a model organism with a short life-cycle that comprises both cellular and multicellular developmental stages. To facilitate studies aimed at detailed knowledge of huntingtins normal function(s), we generated a null mutant of hd, the HD ortholog in Dictyostelium discoideum. Dictyostelium cells lacking endogenous huntingtin were viable but during development did not exhibit the typical polarized morphology of Dictyostelium cells, streamed poorly to form aggregates by accretion rather than chemotaxis, showed disorganized F-actin staining, exhibited extreme sensitivity to hypoosmotic stress, and failed to form EDTA-resistant cell–cell contacts. Surprisingly, chemotactic streaming could be rescued in the presence of the bivalent cations Ca2+ or Mg2+ but not pulses of cAMP. Although hd − cells completed development, it was delayed and proceeded asynchronously, producing small fruiting bodies with round, defective spores that germinated spontaneously within a glassy sorus. When developed as chimeras with wild-type cells, hd − cells failed to populate the pre-spore region of the slug. In Dictyostelium, huntingtin deficiency is compatible with survival of the organism but renders cells sensitive to low osmolarity, which produces pleiotropic cell autonomous defects that affect cAMP signaling and as a consequence development. Thus, Dictyostelium provides a novel haploid organism model for genetic, cell biological, and biochemical studies to delineate the functions of the HD protein.

Loss of Cln3 Function in the Social Amoeba Dictyostelium discoideum Causes Pleiotropic Effects That Are Rescued by Human CLN3

The neuronal ceroid lipofuscinoses (NCL) are a group of inherited, severe neurodegenerative disorders also known as Batten disease. Juvenile NCL (JNCL) is caused by recessive loss-of-function mutations in CLN3, which encodes a transmembrane protein that regulates endocytic pathway trafficking, though its primary function is not yet known. The social amoeba Dictyostelium discoideum is increasingly utilized for neurological disease research and is particularly suited for investigation of protein function in trafficking. Therefore, here we establish new overexpression and knockout Dictyostelium cell lines for JNCL research. Dictyostelium Cln3 fused to GFP localized to the contractile vacuole system and to compartments of the endocytic pathway. cln3− cells displayed increased rates of proliferation and an associated reduction in the extracellular levels and cleavage of the autocrine proliferation repressor, AprA. Mid- and late development of cln3− cells was precocious and cln3− slugs displayed increased migration. Expression of either Dictyostelium Cln3 or human CLN3 in cln3− cells suppressed the precocious development and aberrant slug migration, which were also suppressed by calcium chelation. Taken together, our results show that Cln3 is a pleiotropic protein that negatively regulates proliferation and development in Dictyostelium. This new model system, which allows for the study of Cln3 function in both single cells and a multicellular organism, together with the observation that expression of human CLN3 restores abnormalities in Dictyostelium cln3− cells, strongly supports the use of this new model for JNCL research.

An extracellular matrix, calmodulin-binding protein from Dictyostelium with EGF-like repeats that enhance cell motility

CyrA is a novel cysteine-rich protein with four EGFL repeats that was isolated using the calmodulin (CaM) binding overlay technique (CaMBOT), suggesting it is a CaM-binding protein (CaMBP). The full-length 63kDa cyrA is cleaved into two major C-terminal fragments, cyrA-C45 and cyrA-C40. A putative CaM-binding domain was detected and both CaM-agarose binding and CaM immunoprecipitation verified that cyrA-C45 and cyrA-C40 each bind to CaM in both a Ca(2+)-dependent and -independent manner. cyrA-C45 was present continuously throughout growth and development but was secreted at high levels during the multicellular slug stage of Dictyostelium development. At this time, cyrA localizes to the extracellular matrix (ECM). ECM purification verified the presence of cyrA-C45. An 18 amino acid peptide (DdEGFL1) from the first EGFL repeat sequence of cyrA (EGFL1) that is present in both cyrA-C45 and -C40 enhances both random cell motility and cAMP-mediated chemotaxis. Here we reveal that the dose-dependent enhancement of motility by DdEGFL1 is related to the time of cell starvation. Addition of DdEGFL1 also inhibits cyrA proteolysis. The status of cyrA as an extracellular CaMBP was further clarified by the demonstration that CaM is secreted during development. Antagonism of CaM with W7 resulted in enhanced cyrA proteolysis suggesting a functional role for extracellular CaM in protecting CaMBPs from proteolysis. cyrA is the first extracellular CaMBP identified in Dictyostelium and since it is an ECM protein with EGF-like repeats that enhance cell motility and it likely also represents the first matricellular protein identified in a lower eukaryote.

Calmodulin Binding Proteins and Alzheimer's Disease.

Abstract The small, calcium-sensor protein, calmodulin, is ubiquitously expressed and central to cell function in all cell types. Here the literature linking calmodulin to Alzheimer’s disease is reviewed. Several experimentally-verified calmodulin-binding proteins are involved in the formation of amyloid-β plaques including amyloid-β protein precursor, β-secretase, presenilin-1, and ADAM10. Many others possess potential calmodulin-binding domains that remain to be verified. Three calmodulin binding proteins are associated with the formation of neurofibrillary tangles: two kinases (CaMKII, CDK5) and one protein phosphatase (PP2B or calcineurin). Many of the genes recently identified by genome wide association studies and other studies encode proteins that contain putative calmodulin-binding domains but only a couple (e.g., APOE, BIN1) have been experimentally confirmed as calmodulin binding proteins. At least two receptors involved in calcium metabolism and linked to Alzheimer’s disease (mAchR; NMDAR) have also been identified as calmodulin-binding proteins. In addition to this, many proteins that are involved in other cellular events intimately associated with Alzheimer’s disease including calcium channel function, cholesterol metabolism, neuroinflammation, endocytosis, cell cycle events, and apoptosis have been tentatively or experimentally verified as calmodulin binding proteins. The use of calmodulin as a potential biomarker and as a therapeutic target is discussed.

Aberrant adhesion impacts early development in a Dictyostelium model for juvenile neuronal ceroid lipofuscinosis

ABSTRACT Neuronal ceroid lipofuscinosis (NCL), also known as Batten disease, refers to a group of severe neurodegenerative disorders that primarily affect children. The most common subtype of the disease is caused by loss-of-function mutations in CLN3, which is conserved across model species from yeast to human. The precise function of the CLN3 protein is not known, which has made targeted therapy development challenging. In the social amoeba Dictyostelium discoideum, loss of Cln3 causes aberrant mid-to-late stage multicellular development. In this study, we show that Cln3-deficiency causes aberrant adhesion and aggregation during the early stages of Dictyostelium development. cln3− cells form ∼30% more multicellular aggregates that are comparatively smaller than those formed by wild-type cells. Loss of Cln3 delays aggregation, but has no significant effect on cell speed or cAMP-mediated chemotaxis. The aberrant aggregation of cln3− cells cannot be corrected by manually pulsing cells with cAMP. Moreover, there are no significant differences between wild-type and cln3− cells in the expression of genes linked to cAMP chemotaxis (e.g., adenylyl cyclase, acaA; the cAMP receptor, carA; cAMP phosphodiesterase, pdsA; g-protein α 9 subunit, gpaI). However, during this time in development, cln3− cells show reduced cell-substrate and cell-cell adhesion, which correlate with changes in the levels of the cell adhesion proteins CadA and CsaA. Specifically, loss of Cln3 decreases the intracellular level of CsaA and increases the amount of soluble CadA in conditioned media. Together, these results suggest that the aberrant aggregation of cln3− cells is due to reduced adhesion during the early stages of development. Revealing the molecular basis underlying this phenotype may provide fresh new insight into CLN3 function.

Differentiation inducing factor-1 (DIF-1) induces gene and protein expression of the Dictyostelium nuclear calmodulin-binding protein nucleomorphin

The nucleomorphin gene numA1 from Dictyostelium codes for a multi-domain, calmodulin binding protein that regulates nuclear number. To gain insight into the regulation of numA, we assessed the effects of the stalk cell differentiation inducing factor-1 (DIF-1), an extracellular signalling molecule, on the expression of numA1 RNA and protein. For comparison, the extracellular signalling molecules cAMP (mediates chemotaxis, prestalk and prespore differentiation) and ammonia (NH(3)/NH(4)(+); antagonizes DIF) were also studied. Starvation, which is a signal for multicellular development, results in a greater than 80% decrease in numA1 mRNA expression within 4 h. Treatment with ammonium chloride led to a greater than 90% inhibition of numA1 RNA expression within 2 h. In contrast, the addition of DIF-1 completely blocked the decrease in numA1 gene expression caused by starvation. Treatment of vegetative cells with cAMP led to decreases in numA1 RNA expression that were equivalent to those seen with starvation. Western blotting after various morphogen treatments showed that the maintenance of vegetative levels of numA1 RNA by DIF-1 in starved cells was reflected in significantly increased numA1 protein levels. Treatment with cAMP and/or ammonia led to decreased protein expression and each of these morphogens suppressed the stimulatory effects of DIF-1. Protein expression levels of CBP4a, a calcium-dependent binding partner of numA1, were regulated in the same manner as numA1 suggesting this potential co-regulation may be related to their functional relationship. NumA1 is the first calmodulin binding protein shown to be regulated by developmental morphogens in Dictyostelium being upregulated by DIF-1 and down-regulated by cAMP and ammonia.

Huntingtin regulates Ca2+ chemotaxis and K+-facilitated cAMP chemotaxis, in conjunction with the monovalent cation/H+ exchanger Nhe1, in a model developmental system: Insights into its possible role in Huntington׳s disease

Huntington׳s disease is a neurodegenerative disorder, attributable to an expanded trinucleotide repeat in the coding region of the human HTT gene, which encodes the protein huntingtin. These mutations lead to huntingtin fragment inclusions in the striatum of the brain. However, the exact function of normal huntingtin and the defect causing the disease remain obscure. Because there are indications that huntingtin plays a role in Ca(2+) homeostasis, we studied the deletion mutant of the HTT ortholog in the model developmental system Dictyostelium discoideum, in which Ca(2+) plays a role in receptor-regulated behavior related to the aggregation process that leads to multicellular morphogenesis. The D. discoideum htt(-)-mutant failed to undergo both K(+)-facilitated chemotaxis in spatial gradients of the major chemoattractant cAMP, and chemotaxis up a spatial gradient of Ca(2+), but behaved normally in Ca(2+)-facilitated cAMP chemotaxis and Ca(2+)-dependent flow-directed motility. This was the same phenotypic profile of the null mutant of Nhel, a monovalent cation/H(+)exchanger. The htt(-)-mutant also failed to orient correctly during natural aggregation, as was the case for the Nhel mutant. Moreover, in a K(+)-based buffer the normal localization of actin was similarly defective in both htt(-) and nhe1(-) cells in a K(+)-based buffer, and the normal localization of Nhe1 was disrupted in the htt(-) mutant. These observations demonstrate that Htt and Nhel play roles in the same specific cation-facilitated behaviors and that Nhel localization is directly or indirectly regulated by Htt. Similar cation-dependent behaviors and a similar relationship between Htt and Nhe1 have not been reported for mammalian neurons and deserves investigation, especially as it may relate to Huntington׳s disease.

Reduced amyloidogenic processing of the amyloid β-protein precursor by the small-molecule Differentiation Inducing Factor-1

The detection of cell cycle proteins in Alzheimers disease (AD) brains may represent an early event leading to neurodegeneration. To identify cell cycle modifiers with anti-Abeta properties, we assessed the effect of Differentiation-Inducing Factor-1 (DIF-1), a unique, small-molecule from Dictyostelium discoideum, on the proteolysis of the amyloid beta-protein precursor (APP) in a variety of different cell types. We show that DIF-1 slows cell cycle progression through G0/G1 that correlates with a reduction in cyclin D1 protein levels. Western blot analysis of DIF-treated cells and conditioned medium revealed decreases in the levels of secreted APP, mature APP, and C-terminal fragments. Assessment of conditioned media by sandwich ELISA showed reduced levels of Abeta40 and Abeta42, also demonstrating that treatment with DIF-1 effectively decreases the ratio of Abeta42 to Abeta40. In addition, DIF-1 significantly diminished APP phosphorylation at residue T668. Interestingly, site-directed mutagenesis of APP residue Thr668 to alanine or glutamic acid abolished the effect of DIF-1 on APP proteolysis and restored secreted levels of Abeta. Finally, DIF-1 prevented the accumulation of APP C-terminal fragments induced by the proteasome inhibitor lactacystin, and calpain inhibitor N-acetyl-leucyl-leucyl-norleucinal (ALLN). Our findings suggest that DIF-1 affects G0/G1-associated amyloidogenic processing of APP by a gamma-secretase-, proteasome- and calpain-insensitive pathway, and that this effect requires the presence of residue Thr668.