#242
Gravitropism and vesicular trafficking

Glenn Hicks (P.I.: Natasha Raikhel and Zhenbiao Yang)

Stephanie Robert, Jae-Ung Hwang and Shundai Li

It is known that there are genetic links between gravitropic response and the endomembrane system. Thus, a two-phase screening approach was employed for this screen that took advantage of this connection. The primary screen was for chemicals that affected root gravitropism; whereas the secondary screen examined directly the localization of endomembrane system fluorescent protein markers for altered compartment morphologies. SCREEN DETAILS: For the primary screen a total of 22,000 diverse chemicals were screened. The libraries were: 1) Sigma TimTec Myria Screen (MS) (10,000), 2) Chembridge Nova Core Library (NC) (10,000), and 3) Selected chemicals from Microsource Spectrum (SP). The MS and NC libraries were screened at a concentration of 50-100uM in 400ul of medium using either our custom chemical plates or standard 48-well microtiter plates. Seeds were germinated on media with chemicals. The plates were rotated 90 degrees 3-4 days post-germination and grown for an additional 24hrs. Chemicals that inhibited gravitropism were selected. For the secondary screen, seedlings expressing PIN2-GFP and markers for different endomembrane compartments were germinated and grown for 4-5 days on chemicals at concentrations below those of the primary screen. Overall, only compounds that altered marker localization and inhibited gravitropism were selected. Chemicals that primarily inhibited growth (and thus indirectly inhibited gravitropism) were excluded because they could target non-endomembrane pathways. SUMMARY STATISTICS: Of the 20,000 chemicals screened, a total of 669 (3.4%) (526 from the MS and 143 from the NC libraries) inhibited gravitropism in the primary screen. In the secondary screen, 63 of the 669 chemicals (0.3% of the 20,000) resulted in altered PIN2-GFP localization/expression. Among the 63 effectors of PIN2-GFP localization/expression, 9 inhibited root gravitropism without affecting root growth (0.05% of 20,000). Six of these 9 (0.03% of the 20,000) were confirmed to alter the localization/expression of PIN2-GFP or different endomembrane markers.

NSF: 2010-0520325

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Forward Screenyes Target Pathway

In total, 6 compounds from 2 libraries are annotated. Out of these, 6 have a score of 1 or higher, and 0 are annotated as inactive.

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Screen-level Files

Screen Strategy

Gravitropism (primary), altered localization/expression of PIN2-GFP (secondary)

#15326

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Compound Files

Sigma-TimTec Myria Screen R412333

Library	Sigma-TimTec Myria Screen
ID	R412333
Name
Formula	C17H19NO3
Molecular Weight	285.338

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PIN2-GFP
Assay 1: 2

#117015

Assay 1: Gravitropism and vesicular trafficking

Description PIN2-GFP
Score 2: active in primary and secondary screen
Concentration 25-50uM

PIN2-GFP

25-50uM chemical (left) compared to DMSO control (right). Result: Reduced PIN2-GFP.

#15333

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PIN1-GFP

13-25uM chemical (left) compared to DMSO control (right). Result: Reduced PIN1-GFP.

#15335

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Phototropin1-GFP

25-50uM chemical (left) compared to DMSO control (right). Results: Similar

#15337

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PIP-GFP (plasma membrane)

25-50uM chemical (left) compared to DMSO control (right). Results: Modest mislocalization

#15339

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deltaTIP-GFP(Tonoplast)

25-50uM chemical (left) compared to DMSO control (right). Results: Modest effect

#15350

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Sigma-TimTec Myria Screen ST025823

Library	Sigma-TimTec Myria Screen
ID	ST025823
Name
Formula	C17H19ClN2O2
Molecular Weight	318.798

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PIN2-GFP
Assay 1: 2

#117014

Assay 1: Gravitropism and vesicular trafficking

Description PIN2-GFP
Score 2: active in primary and secondary screen
Concentration 3-5uM

PIN2-GFP

3-5uM chemical (left) compared to DMSO control (right). Results: Reduced PIN2-GFP

#15352

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PIN1-GFP (Plasma membrane)

25-50uM chemical (left) compared to DMSO control (right). Results: Reduced PIN1-GFP

#15354

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PIP (Plasma membrane)

25-50uM chemical (left) compared to DMSO control (right). Results: Similar

#15356

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deltaTIP-GFP (Tonoplast)

13-25uM chemical (left) compared to DMSO control (right). Results: Modest effect

#15358

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Sigma-TimTec Myria Screen R462438

Library	Sigma-TimTec Myria Screen
ID	R462438
Name
Formula	C13H9ClFNO3
Molecular Weight	281.667

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PIN2-GFP
Assay 1: 2

#117016

Assay 1: Gravitropism and vesicular trafficking

Description PIN2-GFP
Score 2: active in primary and secondary screen
Concentration 3-5uM

PIN2-GFP (Plasma membrane)

3-5uM chemical (left) compared to DMSO control (right). Result: Reduced PIN2-GFP

#15360

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PIN1-GFP

3-5uM chemical (left) compared to DMSO control (right). Results: Reduced PIN1-GFP

#15361

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Phototropin1-GFP (Plasma membrane)

25-50uM chemical (left) compared to DMSO control (right). Results: Reduced Phototropin1-GFP

#15362

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PIP-GFP (Plasma membrane)

25-50uM chemical (left) compared to DMSO control (right). Results: Modest mislocalization

#15363

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deltaTIP-GFP (Tonoplast)

25-50uM chemical (left) compared to DMSO control (right). Results: Mislocalization

#15364

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Sigma-TimTec Myria Screen R464481

Library	Sigma-TimTec Myria Screen
ID	R464481
Name
Formula	C9H11N3O3S2
Molecular Weight	273.332

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PIN2-GFP
Assay 1: 2

#117019

Assay 1: Gravitropism and vesicular trafficking

Description PIN2-GFP
Score 2: active in primary and secondary screen
Concentration 25-50uM

PIN2-GFP (Plasma membrane)

25-50uM chemical (left) compared to DMSO control (right). Results: Significant mislocalization

#15365

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PIN1-GFP (Plasma membrane)

25-50uM chemical (left) compared to DMSO control (right). Results: Reduced PIN1-GFP; mislocalization

#15366

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Phototropin1-GFP (Plasma membrane)

25-50uM chemical (left) compared to DMSO control (right). Results: Reduced Phototropin1-GFP

#15367

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PIP (Plasma membrane)

25-50uM chemical (left) compared to DMSO control (right). Results: Similar

#15368

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deltaTIP-GFP (Tonoplast)

25-50uM chemical (left) compared to DMSO control (right)

#15405

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Sigma-TimTec Myria Screen R478814

Library	Sigma-TimTec Myria Screen
ID	R478814
Name
Formula	C17H14Cl2N2O2S
Molecular Weight	381.276

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PIN2-GFP
Assay 1: 2

#117017

Assay 1: Gravitropism and vesicular trafficking

Description PIN2-GFP
Score 2: active in primary and secondary screen
Concentration 13-25uM

PIN2-GFP (plasma membrane)

13-25uM chemical (left) compared to DMSO control (right). Results: Reduced PIN2-GFP

#15406

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PIN1-GFP (Plasma membrane)

25-50uM chemical (left) compared to DMSO control (right). Results: Similar

#15407

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Photropin1-GFP

25-50uM chemical (left) compared to DMSO control (right). Results: Reduced Phototropin1-GFP

#15408

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PIP-GFP (Plasma membrane)

25-50uM chemical (left) compared to DMSO control (right). Results:Mislocalization

#15409

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deltaTIP-GFP (Tonoplast)

25-50uM chemical (left) compared to DMSO control (right)

#15410

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Microsource Spectrum 01500538

Library	Microsource Spectrum
ID	01500538
Name	SPECTINOMYCIN-HYDROCHLORIDE
Formula	C14H26Cl2N2O7
Molecular Weight	405.271

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PIN2-GFP
Assay 1: 2

#117018

Assay 1: Gravitropism and vesicular trafficking

Description PIN2-GFP
Score 2: active in primary and secondary screen
Concentration 25-50uM

PIN2-GFP (plasma membrane)

25-50uM chemical (left) compared to DMSO control (right). Results: Reduced PIN2-GFP

#15411

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PIN1-GFP (Plasma membrane)

25-50uM chemical (left) compared to DMSO control (right). Results: Modest reduction in PIN1-GFP

#15412

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Phototropin1-GFP (Plasma membrane)

25-50uM chemical (left) compared to DMSO control (right). Results: Similar

#15413

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PIP-GFP (plasma membrane)

25-50uM chemical (left) compared to DMSO control (right). Results: Similar

#15414

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deltaTIP-GFP (Tonoplast)

13-25uM chemical (left) compared to DMSO control (right)

#15415

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#242 Gravitropism and vesicular trafficking

Glenn Hicks (P.I.: Natasha Raikhel and Zhenbiao Yang) Stephanie Robert, Jae-Ung Hwang and Shundai Li

Screen-level Files

Compound Files

#242
Gravitropism and vesicular trafficking

Glenn Hicks (P.I.: Natasha Raikhel and Zhenbiao Yang)

Stephanie Robert, Jae-Ung Hwang and Shundai Li