live-cell-cycle-analysis-drosophila-tissues-using-attune-acoustic

Live Cell Cycle Analysis of <em>Drosophila</em> Tissues using the Attune Acoustic Focusing Cytometer and Vybrant DyeCycle Violet DNA Stain

Flow cytometry has  been widely used to obtain information about DNA content in a population of  cells, to infer relative percentages in different cell ...

University of Michigan 

Copines are calcium-dependent membrane-binding proteins found in many eukaryotic organisms. We are studying the function of copines using the model organism, Dictyostelium discoideum. When under starvation conditions, Dictyostelium cells aggregate into mounds that become migrating slugs, which can move toward light and heat before culminating into a fruiting body. Previously, we showed that Dictyostelium cells lacking the copine A (cpnA) gene are not able to form fruiting bodies and instead arrest at the slug stage. In this study, we compared the slug behavior of cells lacking the cpnA gene to the slug behavior of wild-type cells. The slugs formed by cpnA- cells were much larger than wild-type slugs and exhibited no phototaxis and negative thermotaxis in the same conditions that wild-type slugs exhibited positive phototaxis and thermotaxis. Mixing as little as 5% wild-type cells with cpnA- cells rescued the phototaxis and thermotaxis defects, suggesting that CpnA plays a specific role in the regulation of the production and/or release of a signaling molecule. Reducing extracellular levels of ammonia also partially rescued the phototaxis and thermotaxis defects of cpnA- slugs, suggesting that CpnA may have a specific role in regulating ammonia signaling. Expressing the lacZ gene under the cpnA promoter in wild-type cells indicated cpnA is preferentially expressed in the prestalk cells found in the anterior part of the slug, which include the cells at the tip of the slug that regulate phototaxis, thermotaxis, and the initiation of culmination into fruiting bodies. Our results suggest that CpnA plays a role in the regulation of the signaling pathways, including ammonia signaling, necessary for sensing and/or orienting toward light and heat in the prestalk cells of the Dictyostelium slug.

Copine A is expressed in prestalk cells and regulates slug phototaxis and thermotaxis in developing Dictyostelium.

Copines sont d&eacute;pendants du calcium prot&eacute;ines membranaires trouv&eacute;s chez de nombreux organismes eucaryotes. Nous &eacute;tudions la fonction des copines &agrave; l&#39;aide de l&#39;organisme mod&egrave;le, Dictyostelium discoideum. Lorsque, dans des conditions de famine, Dictyostelium cellules agr&eacute;g&eacute;es en monticules qui deviennent des limaces migration, qui peuvent se d&eacute;placer vers la lumi&egrave;re et la chaleur, avant de culminer en un organe de fructification. Auparavant, nous avons montr&eacute; que les cellules de Dictyostelium d&eacute;pourvues de la copine d&#39;un g&egrave;ne (AIIAC) ne parviennent pas &agrave; former des organes de fructification et arr&ecirc;ter &agrave; la place du stade de la limace. Dans cette &eacute;tude, nous avons compar&eacute; le comportement de limace de cellules d&eacute;pourvues du g&egrave;ne AIIAC au comportement de limace de cellules de type sauvage. Les limaces form&eacute;s par AIIAC-cellules &eacute;taient beaucoup plus grands que les limaces sauvage et n&#39;expos&eacute; aucun phototaxie et thermotactisme n&eacute;gatif dans les m&ecirc;mes conditions que les limaces sauvage expos&eacute; thermotactisme et phototaxie positive. M&eacute;lange aussi peu que 5 % des cellules de type sauvage avec AIIAC-cellules ont secouru les d&eacute;fauts phototaxie et thermotactisme, ce qui sugg&egrave;re que l&#39;AIIAC joue un r&ocirc;le sp&eacute;cifique dans la r&eacute;gulation de la production et/ou de la lib&eacute;ration d&#39;une mol&eacute;cule de signalisation. R&eacute;duisant les concentrations extracellulaires d&#39;ammoniac aussi partiellement sauv&eacute; les d&eacute;fauts phototaxie et thermotactisme d&#39;AIIAC-limaces, ce qui sugg&egrave;re que l&#39;AIIAC peut-&ecirc;tre avoir un r&ocirc;le sp&eacute;cifique dans la r&eacute;gulation de la signalisation de l&#39;ammoniac. Exprimant le g&egrave;ne lacZ sous le promoteur AIIAC dans les cellules de type sauvage a indiqu&eacute; AIIAC s&#39;exprime pr&eacute;f&eacute;rentiellement dans les cellules pr&eacute;p&eacute;donculaires ont trouv&eacute; dans la partie ant&eacute;rieure de la limace, qui comprennent les cellules &agrave; l&#39;extr&eacute;mit&eacute; de la limace qui r&eacute;gissent la phototaxie, thermotactisme et l&#39;ouverture de point culminant dans les organes de fructification. Nos r&eacute;sultats sugg&egrave;rent que l&#39;AIIAC joue un r&ocirc;le dans la r&eacute;gulation des voies de signalisation, y compris l&#39;ammoniac signalisation, n&eacute;cessaire &agrave; la d&eacute;tection et/ou l&#39;orienter vers la lumi&egrave;re et la chaleur dans les cellules pr&eacute;p&eacute;donculaires de la limace de Dictyostelium.

Copine A est exprim&eacute;e dans les cellules pr&eacute;p&eacute;donculaires et r&eacute;gule la phototaxie limace et thermotactisme dans le d&eacute;veloppement de Dictyostelium.

Girlfriends sono proteine di membrana-associazione calcio-dipendente trovate in molti organismi eucarioti. Stiamo studiando la funzione di Girlfriends utilizzando l&#39;organismo modello, Dictyostelium discoideum. Quando in condizioni di fame, Dictyostelium cellule aggregate in tumuli che diventano lumache la migrazione, che possono muoversi verso la luce e il calore prima di culminare in un corpo fruttifero. In precedenza, abbiamo dimostrato che cellule di Dictyostelium manca il copine un gene (cpnA) non sono in grado di formare corpi fruttiferi e invece di arresto nella fase slug. In questo studio abbiamo confrontato il comportamento slug di cellule privo del gene cpnA al comportamento del selvaggio-tipo celle slug. Le lumache formate da cellule cpnA erano molto pi&ugrave; grandi di selvaggio-tipo lumache ed non esposti fototassi e termotassi negativo nelle stesse condizioni che lumache selvaggio-tipo esposto termotassi e fototassi positivo. Cos&igrave; piccolo quanto 5% di miscelazione selvaggio-tipo celle con cpnA-celle salvarono i difetti fototassi e termotassi, suggerendo che CpnA svolge un ruolo specifico nel regolamento della produzione e/o il rilascio di una molecola di segnalazione. Riduzione dei livelli extracellulari di ammoniaca anche parzialmente salvato i difetti fototassi e termotassi di cpnA-lumache, suggerendo che CpnA possono avere un ruolo specifico nella regolazione della segnalazione di ammoniaca. Che esprime il gene lacZ sotto il promotore cpnA nel selvaggio-tipo celle indicato cpnA preferenzialmente &egrave; espresso nelle cellule prestalk trovate nella parte anteriore della lumaca, che comprendono le cellule alla punta di slug che regolano la fototassi, termotassi e l&#39;inizio del culmine in corpi fruttiferi. I nostri risultati suggeriscono che CpnA gioca un ruolo nella regolazione delle vie di segnalazione, tra cui ammoniaca segnalazione, necessario per la rilevazione e/o orientare verso la luce e calore nelle cellule prestalk di Dictyostelium slug.

Kerry Flegel

Molecular,
Cellular and Developmental Biology,
Molecular, Cellular and Developmental Biology

University of Michigan

Live Cell Cycle Analysis of Drosophila Tissues using the Attune Acoustic Focusing Cytometer and Vybrant DyeCycle Violet DNA Stain

Kerry Flegel

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}Molecular,Cellular and Developmental Biology,Molecular, Cellular and Developmental Biology

University of Michigan

Live Cell Cycle Analysis of Drosophila Tissues using the Attune Acoustic Focusing Cytometer and Vybrant DyeCycle Violet DNA Stain

Molecular,
Cellular and Developmental Biology,
Molecular, Cellular and Developmental Biology