Name: investigating the function of coronin a in the early starvation response of dictyostelium discoideum by aggregation assays
Uploaded: 2016-06-18T15:00:00
Description: Watch this Scientific Journal Video about Investigating the Function of Coronin A in the Early Starvation Response of Dictyostelium discoideum by Aggregation Assays at JoVE.com

We thank the Dictyostelium Stock Center for strains and reagents. This study was financed by grants from the Swiss National Science Foundation and the Canton of Basel.

<ol><li> Observe a resposta de fome precoce de Dictyostelium discoideum por microscopia de lapso de tempo. <ol><li> Cultivar células DH1.10 ou células cora -deficient num balão de Erlenmayer contendo HL-5 meio (para 1 L: 5 g de proteose peptona, 5 g thiotone E peptona, 10 g de glicose, 5 g de extracto de levedura, 0,35 g de Na 2 HPO 4 * 7H 2 O, 0,35 g de KH 2 PO 4, 0,05 g de sulfato de di-hidroestreptomicina-, pH 6,6) a 22 ° C numa incubadora com agitação ...

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As proteínas coronin são encontrados na maioria dos taxa do clado eucariótica. Dictyostelium discoideum coronin A, o homólogo de coronin mamíferos 1, está envolvida na resposta de fome precoce, uma vez que coronin Um deficiente em células não são capazes de formar centros de agregação durante o início do desenvolvimento ciclo 10. Para ser capaz de avaliar quantitativamente e com precisão o atraso no desenvolvimento entre as cepas, um microscópio imagens de células vivas set-up com o co...

A família de proteínas coronin é altamente conservada durante toda eucariotas. Estas proteínas são caracterizadas pela presença de um triptofano-aspartato amino-terminal (WD) região seguido por uma região única ligado a um domínio em espiral espiralada carboxi-terminal contendo 13,14 repetir-(Figura 1). Coronins têm sido implicados numa variedade de funções celulares, incluindo a regulação do citoesqueleto e transdução de sinal 12. Nos mamíferos, até seis moléculas curtas coronin coronin (1-6), bem como um &quot;conjunto&quot; coronin 7, pode ser co-expressa 12,15. Coronin um membro da família é o mais extensivamente estudado, e foi mostrado para ser envolvido na destruição de agentes patogénicos, a sobrevivência das células T e sinalização neuronal. Como, exatamente, coronin 1 realiza estas actividades ainda não está claro. Enquanto coronin 1 foi mostrado para regular Ca 2+ e de sinalização dependente de cAMP, bem como de F-actina do citoesqueleto modulação 16-18, o potencial de co-expression de até 7 membros da família em mamíferos tornou-se um desafio para estudar a função molecular de coronins nesses sistemas, devido a potenciais despedimentos. Ao contrário de organismos de mamíferos, o discoideum eucariota Dictyostelium inferior expressa apenas dois membros coronin familiares (coronin A, o ortólogo de mamífero coronin 1 e coronin B, ortï¿½logo de coronin mamíferos 7) com funções aparentemente não redundantes 15,19,20. Este fato faz com Dictyostelium discoideum um modelo potente para estudar a função dos coronins. Para estudar o papel de coronin A em Dictyostelium discoideum, que induziu o ciclo de desenvolvimento pela inanição em placas de cultura de tecidos contendo solução de sal equilibrada tampão (BSS) usando quer células do tipo selvagem ou de células sem coronin Um 10. Descobrimos que as células sem coronin A foram incapazes de formar agregados multicelulares sobre fome. Para uma avaliação quantitativa exacta deste fenótipoimagens de células vivas automatizado descrito neste protocolo é uma ferramenta vital. O defeito na iniciação da resposta de fome no início de células sem coronin A pode ser resgatado por fornecimento de impulsos de AMPc, o que sugere que coronin A actua a montante da cascata do AMPc. A aplicação exógena de impulsos de campo para simular a iniciação do desenvolvimento tem sido utilizado por vários laboratórios no passado 8,9. No entanto, este procedimento é também conhecido por ser altamente dependente da densidade das células e de temporização. Portanto, o protocolo aqui descrito destina-se a reduzir estes variabilidades, a fim de garantir um elevado grau de reprodutibilidade. Tomadas em conjunto, as técnicas utilizadas nestes estudos fornecem ferramentas robustas para investigar as funções das proteínas durante as fases iniciais do ciclo de desenvolvimento de Dictyostelium discoideum e tem o potencial para identificar para cima, bem como efectores a jusante de coronin uma função.

<table>
	<tbody>
		<tr>
			<td>HL-5 media (for 1L: 5 g proteose peptone, 5 g thiotone E peptone, 10 g glucose, 5 g yeast extract, 0.35 g Na2HPO4&#8727;2H2O, 0.35 g KH2PO4, 0.05 g dihydrostreptomycin-sulfate, pH 6.6)</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Proteose peptone</td>
			<td>BD Bioscience</td>
			<td>211693</td>
			<td></td>
		</tr>
		<tr>
			<td>Thiotone E peptone</td>
			<td>BD Bioscience</td>
			<td>211684</td>
			<td></td>
		</tr>
		<tr>
			<td>Yeast extract</td>
			<td>BD Bioscience</td>
			<td>212750</td>
			<td></td>
		</tr>
		<tr>
			<td>Glucose</td>
			<td>AppliChem</td>
			<td>A3666</td>
			<td></td>
		</tr>
		<tr>
			<td>Na2HPO4&#8727;2H2O</td>
			<td>Fluka</td>
			<td>71643</td>
			<td></td>
		</tr>
		<tr>
			<td>KH2PO4</td>
			<td>AppliChem</td>
			<td>A1043</td>
			<td></td>
		</tr>
		<tr>
			<td>dihydrostreptomycin-sulfate</td>
			<td>Sigma-Aldrich</td>
			<td>D1954000</td>
			<td></td>
		</tr>
		<tr>
			<td>PBM (0.02 M potassium phosphate, 10 &#956;M CaCl2, and l mM MgCl2, pH 6.1)</td>
			<td></td>
			<td></td>
			<td>self made</td>
		</tr>
		<tr>
			<td>BSS (10 mM NaCl, 10 mM KCl, 2.5 mM CaCl2, pH 6.5)</td>
			<td></td>
			<td></td>
			<td>self made</td>
		</tr>
		<tr>
			<td>0.45-&#956;m Filtropure S filter</td>
			<td>Sarstedt</td>
			<td>83.1826</td>
			<td></td>
		</tr>
		<tr>
			<td>Falcon 24-well Tissue culture plate</td>
			<td>Fisher Scientific</td>
			<td><a href="https://www.fishersci.com/shop/products/falcon-tissue-culture-plates-23/087721h">08-772-1H</a></td>
			<td></td>
		</tr>
		<tr>
			<td>Cellobserver microscope</td>
			<td>Zeiss</td>
			<td></td>
			<td>custom built</td>
		</tr>
		<tr>
			<td>AxioVision software</td>
			<td>Zeiss</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>IPC Microprocessor&#8211;controlled dispensing pump</td>
			<td>ISMATEC</td>
			<td>ISM 931</td>
			<td></td>
		</tr>
		<tr>
			<td>Axiovert 135M microscope</td>
			<td>Zeiss</td>
			<td>491237-0001-000</td>
			<td></td>
		</tr>
		<tr>
			<td>Incubation Shaker</td>
			<td>Inforst HT Minitron</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

investigating the function of coronin a in the early starvation response of dictyostelium discoideum by aggregation assays

Dictyostelium discoideum ameba são encontrados no solo, alimentando-se de bactérias. Quando as fontes de alimentos se tornam escassos, eles secretam fatores para iniciar um programa de desenvolvimento multicelular, durante o qual células individuais chemotax para os centros de agregação 1-4. Este processo é dependente da libertação de monofosfato de adenosina cíclico (cAMP) 5. O AMPc é produzido em ondas através da acção concertada de adenilato-ciclase e fosfodiesterase, e liga-se a receptores de G cAMP acoplados à proteína 6,7. Um ensaio amplamente utilizado para analisar os mecanismos envolvidos no ciclo de desenvolvimento do Dictyostelium discoideum eucariota inferior é baseado na observação de agregação de células em condições submersas 8,9. Este protocolo descreve a análise do papel de coronin A no ciclo de desenvolvimento pela inanição em placas de cultura de tecidos imersos em solução salina equilibrada (BSS) 10. Coronin A é um membro da PR amplamente conservadaotein família de coronins que têm sido implicados numa grande variedade de actividades 11,12. Dictyostelium células que carecem coronin A são incapazes de formar agregados multicelulares, e este defeito pode ser resgatado por fornecimento de impulsos de AMPc, o que sugere que coronin A actua a montante do cAMP cascata 10. As técnicas descritas nestes estudos fornecem ferramentas robustas para investigar as funções das proteínas durante as fases iniciais do ciclo de desenvolvimento de Dictyostelium discoideum a montante da cascata do AMPc. Assim, a utilização deste ensaio de agregação pode permitir a um estudo mais aprofundado da coronin Uma função e fazer avançar nossa compreensão da biologia coronin.

As células deficientes em coronin Um show de um defeito no desenvolvimento inicial (Figura 2). Na ausência de um coronin células são incapazes de formar agregados multicelulares, que é o passo inicial durante o ciclo de desenvolvimento de Dictyostelium discoideum. Portanto, coronin Um parece desempenhar um papel durante a resposta precoce fome e / ou a sinalização de cAMP. Com efeito, a falta de formação de agregados multicelulares na ausência de coron...

Watch this Scientific Journal Video about Investigating the Function of Coronin A in the Early Starvation Response of Dictyostelium discoideum by Aggregation Assays at JoVE.com

Investigating the Function of Coronin A in the Early Starvation Response of Dictyostelium discoideum by Aggregation Assays

The social amoeba Dictyostelium discoideum undergoes a developmental transition into a multicellular organism when starved. The evolutionary conserved protein coronin A plays a crucial role in the initiation of development. Using aggregation assays as our main method, we aim to elucidate the role of coronin A in early development.

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Dictyostelium discoideum amoeba are found in soil, feeding on bacteria. When food sources become scarce, they secrete factors to initiate a multicellular ...

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Investigating the Function of Coronin A in the Early Starvation Response of Dictyostelium discoideum by Aggregation Assays

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Whereas cation transport by the electrogenic membrane transporter Na+,K+-ATPase can be measured by electrophysiology, the electroneutrally operating ...

Live Cell Imaging of Early Autophagy Events: Omegasomes and Beyond

Imagens de células vivas dos primeiros autofagia Eventos: Omegasomes and Beyond

Autophagy is a  cellular response triggered by the lack of nutrients, especially the absence of  amino acids. Autophagy is defined by the formation of ...

Analysis of Skeletal Muscle Defects in Larval Zebrafish by Birefringence and Touch-evoke Escape Response Assays

Zebrafish (Danio rerio) have become a particularly effective tool for modeling human diseases affecting skeletal muscle, including muscular ...

In Vitro Aggregation Assays Using Hyperphosphorylated Tau Protein

In Vitro Aggregation Assays Using Hyperphosphorylated Tau Protein

In Vitro Agregação ensaios utilizando hiperfosforilada Tau Protein

Alzheimer's disease is one of a large group of neurodegenerative disorders known as tauopathies that are manifested by the neuronal deposits of ...

Analysis of Nephron Composition and Function in the Adult Zebrafish Kidney

Análise de Composição Nephron e função no rim adulto Zebrafish

The zebrafish model has emerged as a relevant system to study kidney development, regeneration and disease. Both the embryonic and adult zebrafish kidneys ...

Assessment of Dictyostelium discoideum Response to Acute Mechanical Stimulation

Assessment of Dictyostelium discoideum Response to Acute Mechanical Stimulation

Avaliação de Dictyostelium discoideum resposta à estimulação mecânica aguda

Chemotaxis, or migration up a gradient of a chemoattractant, is the best understood mode of directed migration. Studies using social amoeba Dictyostelium ...