Capture of AT-rich Chromatin by ELYS Recruits POM121

and NDC1 to Initiate Nuclear Pore Assembly

By Rasala et al.

Presented by Laurent PalmatierBGGN 22202/02/10

Introduction

•All eukaryotes have a nuclear envelope (NE)

•Includes nuclear membranes, and large nuclear pore complexes (NPCs) built by 500 – 1000 proteins

The Nucleus

Nuclear pore complex (NPC)

Nuclear membranes

•Nuclear membrane and NPC reformation occurs at the end of mitosis

•This study explores proteins involved in the early NPC reformation

Introduction

•The study is carried out in vitro using Xenopus eggs and sperm for essential biomolecules (proteins, membranes, chromatin)

Synthesis of an ELYS Antibody

•ELYS is known to play an early role in NPC formation

•An antibody stain showed colocalization of ELYS with known nucleoporins (Nups)

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Specificity of ELYS localization

•AL are cytoplasmic membranes with pores

•Immunoblot studies show ELYS purifies with nuclei but not AL

•ELYS likely has a binding affinity for chromatin

How does ELYS bind to chromatin?

•Known to have an AT-hook motif at the C-terminus

•Carried out mutation experiments to verify this region can bind to chromatin

How does ELYS bind to chromatin?

•Immunoblot analysis of three samples shows relative binding affinities

Chromatin Binding and NPC formation

Inhibition of ELYS and Nup107-160 binding to chromatin

AT-hook + / AT-hook 2RA / ΔAT-hook

Determining effects on nuclear pore assembly using anti-FG-nucleoporin antibody: Alexa-568-mAb414

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Chromatin binding and NPC formationAT-hook + / AT-hook 2RA / ΔAT-hook

Are lower Ran-GTP levels responsible for lack of nuclear pore formation?

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Excess Ran-GTP

AT-hook + acts at the surface of the chromatin, inhibiting binding of endogenous ELYS.

Distamycin A and Chromomycin A3

•Antibiotics Distamycin A and Chromomycin A3 bind to AT and GC rich DNA respectively

•Distamycin A blocks nuclear pore assembly, but Chromomycin A3 does not

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Distamycin A blocks by binding to DNA

•AL control shows distamycin A inhibits via DNA binding

•This is because known nucleoporins in the AL are still identified in the presence of distamycin A

Distamycin A inhibits ELYS / chromatin binding

Anchored chromatin assay carried out to definitively show distamycin A blocks ELYS/chromatin binding

Nuclear import assays

Nuclear import assays with GFP-M9 support previous results

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Two Hypotheses:

1) Additional soluble pore subunits recruited to the chromatin/ELYS/ Nup107-160 precursor in a membrane-independent manner

2) One or more integral membrane pore protein(s) with associated membrane vesicle or sheet is recruited.

Identifying the next step in NPC formation

Testing first hypothesis:

1) Additional soluble pore subunits recruited to the chromatin/ELYS/ Nup107-160 precursor in a membrane-independent manner

Identifying the next step in NPC formation

•Tested by anchoring chromatin and immunoblotting for bound proteins

•Found that recruitment of membrane must follow ELYS and Nup107-160 binding to chromatin

POM121 is an integral membrane protein involved in early NPC formation

•Importin α and β bound to POM121, but were removed by RanQ69L-GTP

•Nup107-160 bonds strongly to POM121

•Indicates that POM121 recruitment occurs just after ELYS and Nup107-160 binding to the chromatin

•POM121 is known to function early in NPC formation

•POM121 fragment with removed FG repeats avoids transport receptor binding, but allows soluble nucleoporins to bind.

•Pore subunits containing Nup358, Nup214, Nup155, Nup62, and Nup53 showed no affinity for POM121

POM121 recruitment depends on ELYS and Nup107-160 localization to the chromatin

Distamycin A, and ELYS AT-hook + had been found in this study to block ELYS association to the chromatin

Using anti-POM121-AF488, assays for POM121 in four separate experiments show it will not associate without ELYS being bound previously

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Recruitment of integral membrane proteins gp210 and NDC1

Immunoblot analysis determined that recruitment of NDC1 needs ELYS bound to chromatin, but recruitment of gp210 does not.

Timeline of NPC formation

1) ELYS binds to the AT-rich chromatin at two binding sites

2) Nup107-160 complex associates

3) POM121 and NDC1 are actively recruited

4) The remaining soluble pore subunits are assembled

Summary•ELYS binds to chromatin and targets nuclear pore assembly to the surface of chromosomes as nuclei form at the end of mitosis.

•ELYS is not needed for AL pore formation

•ELYS C-terminus contains two chromatin binding domains (including the AT hook domain)

•Excess ELYS AT-hook + fragment blocks recruitment of ELYS and the Nup107-160 complex, and therefore NPC assembly (mutant or Δ AT-hook domain does not)

•Distamycin A inhibits NPC assembly in the same way though Chromomycin A3 does not

•Besides ELYS, Nup 107-160, and possibly Nup153, the remaining soluble pore subunits require membrane recruitment and fusion to associate with forming NPCs

•POM121 binds to the Nup107-160 complex, showing that its recruitment likely follows ELYS/Nup107-160 chromatin

•Inhibition of ELYS chromatin binding also inhibits the recruitment of POM121 and NDC1

Future Work

Transitioning to in vivo experimentation

Working with human NEs in case pore formation occurs differently

Further characterization of subunit functionalities

Studying yeast which lack ELYS

Studying specific base pairs which bind ELYS

Capture of AT-rich Chromatin by ELYS Recruits POM121

and NDC1 to Initiate Nuclear Pore Assembly

By Rasala et al.

Presented by Laurent PalmatierBGGN 22202/02/10