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WHAMM Is an Arp2/3 Complex Activator That Binds Microtubules and Functions in ER to Golgi Transport. Kenneth G. Campellone,1,* Neil J. Webb,1 Elizabeth A. Znameroski,1 and Matthew D. Welch1,* 1Department of Molecular and Cell Biology, University of California, Berkeley, CA 94720, USA
Kenneth G. Campellone,1,* Neil J. Webb,1 Elizabeth A. Znameroski,1 and Matthew D. Welch1,*
1Department of Molecular and Cell Biology, University of California, Berkeley, CA 94720, USA
WHAMM (WASP homolog NPF associated with actin, membranes and microtubules) is nuclear promoting factor specific to vertebrates that regulates the activity of the Arp2/3 complex.
1) regulates ER to Golgi transport
2) interacts with both the actin and microtubule cytoskeletons to control membrane tubulation and dynamics at the Golgi apparatus.
(1.) Class 1 Wiskott-Aldrich Syndrome Protein (WASP) family.
- N-WASP homolog and relatives Scar/WAVE1-3 have broad expression.
(2.) Class II is cortactin
- Acidic region interacts with Arp2/3 to bind filaments.
--WHAMM has two WH2 domains and a tryptophan residue in acidic region.
WHAMM is an Actin Nucleation Promoting FactorQuestion posed: What other activators of Arp2/3 complex are present in the human genome?Results: C-terminal WCA domain with 2 putative WH2 peptides and tryptophan residue (W807). : Polyproline region predicted to bind profilin : Coiled-coil central domain : N-terminus has unspecific homology to proteins in database.WHAMM is <20% similar to Class I NPFs.
WHAMM is expressed in mammalian tissue.
Result: WHAMM is an NPF.
: Full length WHAMM is not tightly autoinhibited, like WCA domain.
WHAMM is an Actin NPFResult: W807A in both FL and WCA WHAMM decreased NPF activity. -Increased time to reach ½ max actin polymerization -Decreased filament elongation rates. :WHAMM shares characteristics with WAVE2 NPF activity, but both are less active than N-WASP.
Question: What’s the cellular function of WHAMM? Where is it expressed?Result: Most highly expressed in brain tissue. It’s expressed ubiquitously!
WHAMM Associates with MembranesQuestion: Due to the fact that NPFs can function near cell membranes, how does WHAMM associate with membranes?Result: WHAMM present more in membrane fraction.
WHAMM associates with Golgi Membranes and Microtubules Question: Does WHAMM localize to the plasma membrane or to internal membranes?Result: Localizes to perinuclear space near MTOC. : Localizes to tubulo-vesicular structures in cell periphery that colocalized along microtubules.
Question: The Golgi apparatus is near the MTOC. Does WHAMM colocalize with other markers for Golgi subcompartments?Result: WHAMM colocalized with cis-Golgi protein GM130. : WHAMM redistributes into cytoplasmic puncta that colocalize with GM130 when treated with nocodazole.
WHAMM associates with Golgi and ERGIC Membranes and Microtubules Question: WHAMM localizes along tubulo-vesicular structures, so does WHAMM associate with membrane transport intermediates (microtubules) that move between ER and Golgi?Result: WHAMM and ERGIC-GFP colocalize along peripheral tubules. : WHAMM-associated tubulo-vesicular membranes are ER-Golgi intermediates.
WHAMM localizes to Golgi Membrane, along MTs at MTOC, along tubular MT membranes. Question: Where does WHAMM localize- membrane or in cytosol?Result: LAP-WHAMM is enriched on membranes. Overexpressed LAP-WHAMM had increased cytosolic levels.
Question: How does WHAMM localization compare to that of other NPF’s?Result: WHAMM is the only NPF specifically associated with Golgi and ERGIC membranes and microtubules.
Question: In what molecular manner does WHAMM bind to Golgi membrane and MTs?Result: GFP-N colocalized with GM130 protein. : GFP-CC colocalized along MTs and in nucleus. Sustained binding caused MT bundling. : GFP-WCA had diffused localization, caused F-actin accumulation
Question: N-terminus and CC domain localize to Golgi membranes and MT’s, so how do the domains bind to membranes in vitro?Result: N terminus mediates membrane interaction.
WHAMM has demonstrated that it has NPF activity, so it can stimulate Arp2/3. It also localizes to the Golgi and along tubular membranes. Can WHAMM stimulate Arp2/3 at the Golgi complex and along tubular membranes?
Question: Does WHAMM associate with Arp2/3?
Result: Arp3 co-precipitates with LAP-WHAMM. W807A did not co-precipitate well.
*WHAMM interacts with Arp2/3 complexes.
Question: Where does WHAMM activate Arp2/3-- at the Golgi or along tubules?Result: Increased F-actin at the Golgi. F-actin & Arp3 along tubular structures.
Comparison of WHAMM’s ability to nucleate actin to other NPFs:
-- Increased F-actin content in GFP-WHAMM and GFP-WAVE2 cells.
-- GFP-WHAMM-WCA mutant did not increase F-actin content, so WCA needed for actin polymerization.
Result: InNS siRNAs LAP-WHAMM and W807A mutant were enriched at Golgi, but W807A enriched F-actin 70% less.
*WCA domain and Arp2/3 must interact for actin polymerization.
ERGIC, cis and trans Golgi networks redistributed with WHAMM overexpression.
Overexpressing other NPFs does not affect Golgi distribution.
WHAMM’s N-Terminus and CC region are mediators for Golgi reorganization in WHAMM overexpressing cells.
Question: Does WHAMM depletion affect Golgi positioning and structure?Results: WHAMM is important regulator of Golgi positioning and structure.
WHAMM Overexpression or Depletion Inhibits Anterograde Transport of VSV-GQuestion: Does WHAMM function in membrane trafficking and transport between ER and Golgi?Results: WHAMM is involved in VSV-G transport from ER to Golgi.
B.) Question: How does WHAMM overexpression affect VSV-G transport?
Results: WT WHAMM and W807A mutant inhibit VSV-G trafficking. Arp2/3 activation is not necessary for vesicle transport.
Result: shWHAMM plasmids caused a decrease in VSV-G transport in Golgi (40% after 15 min) and a decrease in plasma membrane localization (50% reduction).
Result: siWHAMM-treated cells did not exhibit Golgi-like VSV-G fluorescence.
:VSV-G proteins seen in Golgi puncta structures were dispersed.
Level of WHAMM silencing was proportional to reduced transport.
Overexpression and depletion studies reveal WHAMM’s role in regulating ER to Golgi transport, and they demonstrate that this function does not require NPF activity.
WHAMM Tubule Dynamics Requires Interactions with Microtubules and Actin FilamentsQuestion: Does WHAMM mediate cytoskeletal crosstalk during membrane movement?Result: (A) WHAMM is visible on spherical vesicles and on tubulo-vesicular structures.(B) GFP-WHAMM colocalized with (1) ERGIC tubulo-vesicular membranes (2) microtubules (3) tubulo-vesicular membranes that recruited mCherry-actin and elongated.
WHAMM Tubule Dynamics Require Interactions with Microtubules and Actin Filaments Question: What role do MTs play in membrane dynamics?Results: MTs and their motors are required for vesicle movement in addition to membrane tubule formation, elongation and stability.
WHAMM Tubule Dynamics Require Interactions with Microtubules and Actin Filaments Question: What is actin’s role in vesicle and tubule movements? Result: Actin filaments are necessary for elongation and stabilization of WHAMM-associated tubular structures.
WHAMM Tubule Dynamics Require Interactions with Microtubules and Actin Filaments Question: What is the role of WHAMM’s NPF activity in membrane dynamics?Result: WHAMM WCA domain enables efficient tubule elongation by triggering Arp2/3 mediated actin assembly.
1.) WHAMM is a multifunctional protein with modular domain structure:
--the ERGIC and Golgi membrane (N-terminus)
--binds and organizes microtubules (CC domain)
--binds and stimulates Arp2/3-mediated actin polymerization (WCA domain)
2.) Localizes to the perinuclear region, cis-Golgi membrane and ERGIC membrane.
--WHAMM’s presence and protein levels control the localization of the compact and stacked Golgi structure near the MTOC.
3.) WHAMM’s association with MTs and actin adds to the secretory pathway’s efficiency, as its presence and protein level affect anterograde transport.
-WHAMM-associated tubular membranes were transport intermediates directed from ER to cis-Golgi.
-NPF activity not needed for transport.
4.) It interacts with both actin and microtubule cytoskeletons to control membrane tubulation and dynamics
*WHAMM provides insight into the mechanisms through which membrane dynamics with multiple cytoskeleton networks are controlled in mammalian cells.*