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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.

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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 protein

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 protein

Hela

yeast

also see movieS2

Dippold et al., 2009



Golph3 binds pi 4 p via a positively charged pocket1
GOLPH3 Binds PI(4)P via a Positively Charged Pocket protein

Phosphatidylethanolamine

Binding pocket mutants

Dippold et al., 2009


Summary i
Summary I protein

  • 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 proteinAnterograde 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 protein

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??? PI(4)P

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 PI(4)PActin 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 PI(4)P

Hela whole cell lysate

Dippold et al., 2009



Rescue of myo18a knockdown requires an intact myo18a atpase pocket
Rescue of MYO18A Knockdown Requires an intact MYO18A PI(4)PATPase Pocket

Dippold et al., 2009


Pi 4 p golph3 myo18a f actin

Summary II PI(4)P

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 PI(4)P-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- PI(4)PActin 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- PI(4)PActin 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 PI(4)P

  • 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

?

?

?

  • 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.