Juan wu bmmb 12 04 09
This presentation is the property of its rightful owner.
Sponsored Links
1 / 24

Juan Wu BMMB 12/04/09 PowerPoint PPT Presentation


  • 95 Views
  • Uploaded on
  • Presentation posted in: General

Juan Wu BMMB 12/04/09. Golgi Apparatus. http://micro.magnet.fsu.edu/cells/golgi/golgiapparatus.html. PtdIns (4)P/GOLPH3/MYO18A/F- actin apparatus. Apply tensile force that pulls on trans-Golgi membrane to produce the flattened-shaped cisternae.

Download Presentation

Juan Wu BMMB 12/04/09

An Image/Link below is provided (as is) to download presentation

Download Policy: Content on the Website is provided to you AS IS for your information and personal use and may not be sold / licensed / shared on other websites without getting consent from its author.While downloading, if for some reason you are not able to download a presentation, the publisher may have deleted the file from their server.


- - - - - - - - - - - - - - - - - - - - - - - - - - E N D - - - - - - - - - - - - - - - - - - - - - - - - - -

Presentation Transcript


Juan wu bmmb 12 04 09

Juan Wu

BMMB

12/04/09


Golgi apparatus

Golgi Apparatus

http://micro.magnet.fsu.edu/cells/golgi/golgiapparatus.html


Ptdins 4 p golph3 myo18a f actin apparatus

PtdIns(4)P/GOLPH3/MYO18A/F-actinapparatus

  • Apply tensile force that pulls on trans-Golgi membrane to produce the flattened-shaped cisternae.

  • Efficient vesicle budding and transporting from Golgi TGN to PM

Dippold et al., 2009


Ptdins 4 p also named pi 4 p most abundant phospholipid at golgi membrane

PtdIns(4)P (also named PI(4)P): most abundant phospholipid at Golgi membrane

Phosphatidylinositol

Metabolic cycle of PtdIns(4)P in mammals

Matteis et al.,


Lipid binding screen identifies golph3 as a pl 4 p binding protein

Lipid-binding Screen Identifies GOLPH3 as a Pl(4)P-binding protein

IVT system

Bacterial purified

Dippold et al., 2009


Golph3

GOLPH3

A

B PI(4)P reporter EYFP-FAPP1-PH (Godi, et al 2004) colocalize with mCherry-GOLPH3 over space and time. MS1

Dippold et al., 2009


Pi 4 p is required for golph3 localization to the golgi

PI(4)P is Required for GOLPH3 Localization to the Golgi

Hela

yeast

also see movieS2

Dippold et al., 2009


Golph3 binds pi 4 p via a positively charged pocket

GOLPH3 Binds PI(4)P via a Positively Charged Pocket

Dippold et al., 2009


Golph3 binds pi 4 p via a positively charged pocket1

GOLPH3 Binds PI(4)P via a Positively Charged Pocket

Phosphatidylethanolamine

Binding pocket mutants

Dippold et al., 2009


Summary i

Summary I

  • GOLPH3 binds tightly, specifically and directly to PI(4)P in two assays. (Lipid blot and vesicle binding)

  • Binding is evolutionarily conserved from yeast to humans

  • Depletion or blocking PI(4)P results in dissociation of GOLPH3 from Golgi TGN

  • GOLPH3 requires the ability to bind PI(4)P to localize to the Golgi.


Golph3 is required for anterograde trafficking from golgi to pm

GOLPH3 is Required for Anterograde Trafficking from Golgi to PM

Ts045-

Dippold et al., 2009


Golph3 is required for the normal extended golgi ribbon

GOLPH3 is Required for the Normal Extended Golgi Ribbon

Examinnation of several Golgi markers in GOLPH3 knockdown cells:

GM130 (cis-Golgi), glycosyltransferases( cis/medial-Golgi), P130 &TGN46( trans-Golgi) in Hela, HEK293, NIH 3T3 cells are revealed condensation of Golgi.

Dippold et al., 2009


Rescue of golph3 knockdown requires the ability to bind pi 4 p

Rescue of GOLPH3 Knockdown Requires the Ability to Bind PI(4)P

Asterisks denotes transfected cells

Dippold et al., 2009


Juan wu bmmb 12 04 09

Why does Golgi Change Morphology upon GOLPH3 Depletion???

Actin?

Microtubule?

GOLPH3

PI(4)P

GOLPH3

X

G O L G I

LatB treatment led to rapid Golgi condensation

MOVIES3-Live Image


Golph3 links the golgi to the actin cytoskeleton

GOLPH3 Links the Golgi to the Actin Cytoskeleton

  • Knockdown of GOLPH3 results in condensation of Golgi without altering F-actin

  • Depolymerization of the actin by LatB leads to compaction of Golgi without affecting

  • the Golgi association of GOLPH3

Dippold et al., 2009


Golph3 binds myo18a directly

GOLPH3 Binds MYO18A Directly

Hela whole cell lysate

Dippold et al., 2009


Myo18a depends on golph3 for golgi localization

MYO18A depends on GOLPH3 for Golgi Localization

Dippold et al., 2009


Rescue of myo18a knockdown requires an intact myo18a atpase pocket

Rescue of MYO18A Knockdown Requires an intact MYO18A ATPase Pocket

Dippold et al., 2009


Pi 4 p golph3 myo18a f actin

Summary II

PI(4)P/GOLPH3/MYO18A/F-Actin

GOLPH3

p230

Depletion of PI(4)P

P230

Knockdown of GOLPH3

Knockdown of MYO18A

Depolymerization of action

Dippold et al., 2009


Trans golgi cisternae are dilated in cells depleted of golph3 or myo18a

Trans-Golgi Cisternae Are Dilated in Cells Depleted of GOLPH3 or MYO18A

Dippold et al., 2009


Pi 4 p golph3 myo18a f actin are required for efficient vesicle budding

PI(4)P/GOLPH3/MYO18A/F-Actin Are Required for Efficient Vesicle Budding

Live Time-lapse images of EYFP-FAPP1-PH tubules or vesicles leaving Golgi. movie4

Dippold et al., 2009


Pi 4 p golph3 myo18a f actin are required for efficient vesicle budding1

PI(4)P/GOLPH3/MYO18A/F-Actin Are Required for Efficient Vesicle Budding

The initial trajectory of vesicles exit follows the orientation of the tensile force.

Dippold et al., 2009


Summary

Summary

  • GOLPH3 binds to PI(4)P and depends on PI(4)P for its Golgi localization

  • PI(4)P/GOLPH3/MYO18A/F-actin apparatus is required for maintain Golgi flattened cisternae morphology at trans-face.

  • PI(4)P/GOLPH3/MYO18A/F-actin apparatus is also required for efficient vesicle budding and trafficking from Golgi to PM


Future perspectives

Future Perspectives…

?

?

?

  • PI(4)P GOLPH3 MYO18A F-actin

  • How does this molecular overcomes the problem of actin being constantly polymerizing and depolymerizing?

  • How does this molecular apparatus balance between maintaining Golgi morphology and extracting vesicles?

  • How are the flattened cis-and medial-cisternae morphology maintained?

  • Can the similar molecular apparatus be extended to other organelle, like ER?

  • Scott et al., identified GOLPH3 as an oncogene.


  • Login