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Competition and Synergy of Arp2/3 and Formins in Nucleating Actin Waves

Xiang Le Chua, Chee San Tong, X.J. Xŭ, Maohan Su, Shengping Xiao, Xudong Wu, Min Wu

Preprint posted on 13 September 2023 https://www.biorxiv.org/content/10.1101/2023.09.13.557508v1.full

Actin polymerization, wave after wave

Selected by Roberto Amadio

Categories: cell biology

 

Background. Actin dynamics play fundamental roles in various cell biological aspects, ranging from cell division, motility, as well as growth. A peculiar phenomenon of cycling rounds of actin polymerization and disassembly, referred as actin waves, is known to occur in many cell types and has been linked to several processes like migration and plasma membrane rearrangements (Inagaki and Katsuno, 2017). Actin polymerization is orchestrated by two main classes of nucleators, namely Formins and the Arp2/3 complex, which respectively drive elongation of preexisting filaments and formation of new actin branches (Lappalainen et al. 2022). After being described and studied separately for many years, Formins and Arp2/3 are increasingly recognized to work in concert in many circumstances. Still, how their functions are coordinated represent an active area of investigation. In this preprint, the authors employ actin waves as a model to study the interplay between Formins and Arp2/3 in Mast cells. Overall, they found a previously unrecognized synergy between the two classes of nucleators, as well as a competition for the common upstream regulator Cdc42.

 

 

Key findings of this preprint

  1. Formins precede Arp2/3 in coordinated actin waves

The authors used super-resolution microscopy and various fluorescent reporters for the Formin mDia3 and FMNL1 and the Arp2/3 complex in order to catch a potential oscillatory and coordinated movement of these factors at the plasma membrane. Arp2/3 was recruited in all actin waves analyzed and in a punctate pattern, highlighting its importance and specialized function in this context. When present in the same cortical actin wave, Arp2/3 recruitment was consistently seen to lag behind Formins in the cycling steps of actin waves (Figure 1). The authors suggest that Formin-mediated actin polymerization could act as a trigger by providing preexisting filaments where Arp2/3 could be recruited to nucleate new branches.

Figure 1 – Live imaging of Formin FMNL1 and Arp3 at cortical waves.

  1. Molecular mechanism of Formins recruitment to waves

To grasp the mechanism of Formin recruitment to the plasma membrane, the authors overexpressed various fluorescent Formin mutants and found that the GTPase binding domain, together with the releasement of the autoinhibited form of the protein are necessary to timely recruit Formins before Arp2/3 and in a rhythmic fashion (Figure 2).

Figure 2 – Representative picture of kymographs showing actin waves of a constitutively active FMNL1 and a C-terminal FMNL1 portion together with Arp2/3. Releasement from autoinhibition (left) results in well-defined and rhythmic FMNL1 waves, whereas mutation in the GTPase binding domain (right) results in abrogation of a defined pattern of Formin wave.

 

  1. Requirements for actin nucleator recruitment to waves

Blocking actin dynamics by cytochalasin-D treatment allowed the authors to uncouple actin oscillation from membrane recruitment of actin-nucleator factors. Only the Formins and Arp2/3 upstream regulator Cdc42 was able to maintain membrane recruitment in this condition, whereas waves of Arp2/3, FMNL1 or mDia3 were all abolished, highlighting their need for interaction with actin.

 

  1. Cdc42 competition rather than actin competition dictates waves coordination

Blockade of Arp2/3 activity by CK-666 treatment increased Formin and Cdc42 activity and recruitment to cortical waves. Arp2/3 recruitment dampened Formin-mediated actin polymerization, likely by competing for Cdc42 binding. Indeed, Cdc42 can bind both FMNL1 and WASp (the factor guiding Arp2/3 activity downstream of Cdc42) in a mutually exclusive way. Moreover, Arp2/3 activity was observed to somehow downregulate Cdc42 activity. The authors thus suggested that Arp2/3 recruitment functions as negative feedback to Formin at every cycle of an actin wave by competing for Cdc42 binding and activity.

 

 

Why I chose this preprint

Regulation of actin dynamics is a fascinating aspect in cell biology with lots of unanswered questions. The authors used very nice microscopy techniques to get further insights of this process. The evidence provided by the authors challenges the current simplistic view depicting competition between Formins and Arp2/3 for monomer actin and add another layer of complexity by showing Cdc42 as a critical regulator of actin nucleator activities.

 

Questions to the authors

  • Would it be possible to generate fluorescent labelled knock-in to visualize formins and Arp2/3 endogenous localization and activities?
  • How could Arp2/3 activity restrain Cdc42 function?
  • What has been the most challenging part and what the most exciting part of your work for this story?

 

 

References

  • Inagaki N., Katsuno H. “Actin Waves: Origin of Cell Polarization and Migration?” (2017), Trends in Cell Biology, 27 (7), pp. 515-526.
  • Lappalainen, P., Kotila, T., Jégou, A. et al. “Biochemical and mechanical regulation of actin dynamics”. (2022), Nat Rev Mol Cell Biol 23, 836–852. https://doi.org/10.1038/s41580-022-00508-4

 

 

Tags: actin, actin waves, arp2/3, formins

Posted on: 27 October 2023 , updated on: 8 November 2023

doi: https://doi.org/10.1242/prelights.35841

Read preprint (No Ratings Yet)

Author's response

Xiang Le Chua shared

– Would it be possible to generate fluorescent labelled knock-in to visualize formins and Arp2/3 endogenous localization and activities?

The endogenous labeling of Arp2/3 is possible, and it has been showcased in the literature. However, mammalian cells generally express very low levels of formins, therefore the endogenous labeling of formins can be a difficult task to do.

 

– How could Arp2/3 activity restrain Cdc42 function?

The relationship between the Arp2/3 complex and Cdc42 can be complex, and there are several potential mechanisms that we speculate, can restrain Cdc42 function. The Arp2/3 activity can possibly influence Cdc42’s localization and function by affecting the physical and chemical environment. Firstly, Arp2/3-dependent actin polymerization generates a dense branched actin network that can restrict the diffusion of Cdc42 and physically block its access to specific sites on the membrane and downstream effectors, hence limiting its activity and function. Secondly, Arp2/3 and Cdc42 may compete for binding sites on actin-binding proteins such as WASP and N-WASP. When Arp2/3 activity is high, it can outcompete Cdc42, thereby restraining its function. Thirdly, while the Arp2/3 complex and formins typically operate under separate pathways, but they share a common activator, Cdc42. Therefore, the Arp2/3 complex could limit the amount of free active Cdc42 on the plasma membrane, thus impacting formin activation.

 

– What has been the most challenging part and what the most exciting part of your work for this story?

The most challenging aspect has been mitigating the effects of phototoxicity during live-cell imaging. We are passionate about understanding the intricate crosstalk between formins and the Arp2/3 complex, a process which happens at relatively high temporal scale. To capture this, we need to acquire TIRFM images at a relatively rapid pace, typically 1-4 seconds per frame, over prolonged duration to delve into their dynamics. It is challenging yet exciting to be able to unveil the dynamic assembly of different nucleators in real time with high temporal resolution.

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