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 *Correspondence: (K.G.C.), (M.D.W.) DOI /j.cell Katherine Oakley

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. It differs from other NPFs in the following ways: 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.

Arp2/3 nucleates filaments, and it is tightly regulated by NPFs Mammalian protein family NPFs: (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. NPF function resides in C-terminal WCA domain that consists of WASP homology 2 (WH2) peptides that bind actin monomers, and connector & acidic (CA) segments that interact with Arp2/3 complex.

WHAMM as a novel NPF compared to WASPs, WAVEs & cortactin NPF activities of WASPs are lower than WAVEs because WASP WCA regions are autoinhibited by intramolecular interactions with GTPase-binding domain. WAVEs are not autoinhibited. WASPs and WAVEs regulate actin assembly at plasma membrane. WHAMM is similar to class I NPFs, because they stimulate actin nucleation in the same manner. --WHAMM has two WH2 domains and a tryptophan residue in acidic region. WHAMMs NPF activity is more similar to WAVEs than WASPs, because WHAMM is constitutively active in vitro and triggers actin assembly when expressed in cells. It is NOT autoinhibited, like WASPs are.

WHAMM is an Actin Nucleation Promoting Factor Question 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.

Question: What is WHAMMs function in cells? WHAMM is expressed in mammalian tissue.

WHAMM is an Actin Nucleation Promoting Factor Question: WHAMM has a WCA domain, so does it promote actin nucleation? Result: WHAMM is an NPF. : Full length WHAMM is not tightly autoinhibited, like WCA domain.

WHAMM is an Actin NPF Result: 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: Whats the cellular function of WHAMM? Where is it expressed? Result: Most highly expressed in brain tissue. Its expressed ubiquitously!

WHAMM Associates with Membranes Question: 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 NPFs? 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 MTs, so how do the domains bind to membranes in vitro? Result: N terminus mediates membrane interaction.

Question: Which domain of WHAMM binds to microtubules? Result: GST-CC binds microtubules at all molar ratios of them.

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 WHAMMs 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.

Question: How does WHAMM WCA domain interact with Arp2/3? Result: In NS 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.

WHAMM Overexpression or Depletion Disrupts Golgi Apparatus Structure ERGIC, cis and trans Golgi networks redistributed with WHAMM overexpression. Overexpressing other NPFs does not affect Golgi distribution. WHAMMs 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-G Question: 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.

Question: What is WHAMMs role in 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).

Question: How does WHAMM mediate transport from ER to Golgi? 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. Without WHAMM, VSV-G stays in the ER. Overexpression and depletion studies reveal WHAMMs 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 Filaments Question: 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 actins 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 WHAMMs NPF activity in membrane dynamics? Result: WHAMM WCA domain enables efficient tubule elongation by triggering Arp2/3 mediated actin assembly.

Summary 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. --WHAMMs presence and protein levels control the localization of the compact and stacked Golgi structure near the MTOC.

Summary, cont. 3.) WHAMMs association with MTs and actin adds to the secretory pathways 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 Microtubules and motors are required for vesicle transport in addition to initiating and stabilizing membrane tubule formation ER to Golgi membranes (in vitro). WHAMM-associated actin nucleation along ERGIC and cis-Golgi membranes stabilizes the tubulated membranes and facilitates their elongation. *WHAMM provides insight into the mechanisms through which membrane dynamics with multiple cytoskeleton networks are controlled in mammalian cells.*

Future directions Determine interrelationship of actin and microtubule based cytoskeletons and components of the secretory pathway. Further define role for WHAMM during vesicular transport. Does the WHAMM-activated actin assembly provide tension along Golgi membranes to facilitate movement and tubulation? Does it bind COPII coat protein that is found in ER exit sites? If so, how? WHAMMs effect on Golgi to cell surface trafficking? Signaling factor to regulate WHAMM-mediated Arp2/3 activation? Single or combination of GTPase families (Sar, Rab, Arf) that regulate WHAMMs membrane- binding, microtubule-binding or NPF activities. Is it similar to Cdc42 and Rac regulating WASPs and WAVEs? Binding partners to WHAMM that allow it to be constitutively active/not autoinhibited to trigger actin assembly? WHAMMs overexpression defects ER to cis-Golgi transport, so are secretory components mislocalized or is retrograde transport stimulated? Which proteins in other organisms carry out WHAMM-like activities & what are their mechanisms of membrane transport? Does WHAMM coordinate neuronal extension and signaling (as Cobl does) since its expressed highly in brain tissues?