Publishing protocols as a new PI.

Effie Bastounis recently started a lab at the University of Tübingen that studies how physical forces guide the interactions of host cells with bacterial pathogens. Former STAR Protocols Lead editor Shawnna Buttery discussed with Effie her experience publishing research at Cell Press journals and how that led to her publishing in STAR Protocols. Effie also shared her thoughts on the usefulness of protocols journals and the importance of protocols to a new PI. For more information on the protocols related to this backstory, please refer to Muenkel et al.1 and Bastounis et al.2.

How to expand the method details in your Cell Press paper with step-by-step STAR Protocols.

Publishing a primary research article is typically the result of a collaborative effort between a variety of researchers across differing career stages. STAR Protocols can complement a research article and empower authors to share the expertise they contributed to the larger study. In this Backstory, we interview members of the Gennarino lab, who published a Cell paper and four protocols, covering bioinformatics, culturing of patient-derived cell lines, neuroimaging from mouse brain sections and primary neurons, and mouse seizure recordings. For more information on the protocols related to this backstory, please refer to (Gennarino et al., 2018).

Why STAR Protocols authors make excellent protocol reviewers.

When researchers submit a protocol for peer review and publication, they receive feedback from reviewers to help improve the usability of the protocol. These authors can be the perfect peer reviewers helping propel research forward. They can use their technical expertise and sharpened writing skills to help improve the main aspects of published protocols, namely their clarity and reproducibility. This backstory chronicles the journey of Dr. Guillaume Blot, from a junior researcher and author to a protocol reviewer. For complete details, please refer to Blot et al. (2021).

How peer review and publication can make a good protocol even better.

A guiding principle of STAR Protocols is that we make researchers' lives easier by publishing robust and usable protocols. We leverage the strength of peer review to help authors improve their protocol. This Backstory details the transformation of a bench protocol to a published protocol, highlighting the improvements to the article through the drafting, review, and revision stages. This underscores the value of the peer review process in general and the collaborative peer review philosophy at STAR Protocols specifically. For complete details, please refer to Chhoy et al. (2021).

Regulation of the formin Bnr1 by septins anda MARK/Par1-family septin-associated kinase.

Formin-family proteins promote the assembly of linear actin filaments and are required to generate cellular actin structures, such as actin stress fibers and the cytokinetic actomyosin contractile ring. Many formin proteins are regulated by an autoinhibition mechanism involving intramolecular binding of a Diaphanous inhibitory domain and a Diaphanous autoregulatory domain. However, the activation mechanism for these Diaphanous-related formins (DRFs) is not completely understood. Although small GTPases play an important role in relieving autoinhibition, other factors likely contribute. Here we describe a requirement for the septin Shs1 and the septin-associated kinase Gin4 for the localization and in vivo activity of the budding yeast DRF Bnr1. In budding yeast strains in which the other formin, Bni1, is conditionally inactivated, the loss of Gin4 or Shs1 results in the loss of actin cables and cell death, similar to the loss of Bnr1. The defects in these strains can be suppressed by constitutive activation of Bnr1. Gin4 is involved in both the localization and activation of Bnr1, whereas the septin Shs1 is required for Bnr1 activation but not its localization. Gin4 promotes the activity of Bnr1 independently of the Gin4 kinase activity, and Gin4 lacking its kinase domain binds to the critical localization region of Bnr1. These data reveal novel regulatory links between the actin and septin cytoskeletons.

A Ser/Thr kinase required for membrane-associated assembly of the major sperm protein motility apparatus in the amoeboid sperm of Ascaris.

Leading edge protrusion in the amoeboid sperm of Ascaris suum is driven by the localized assembly of the major sperm protein (MSP) cytoskeleton in the same way that actin assembly powers protrusion in other types of crawling cell. Reconstitution of this process in vitro led to the identification of two accessory proteins required for MSP polymerization: an integral membrane phosphoprotein, MSP polymerization-organizing protein (MPOP), and a cytosolic component, MSP fiber protein 2 (MFP2). Here, we identify and characterize a 34-kDa cytosolic protein, MSP polymerization-activating kinase (MPAK) that links the activities of MPOP and MFP2. Depletion/add-back assays of sperm extracts showed that MPAK, which is a member of the casein kinase 1 family of Ser/Thr protein kinases, is required for motility. MPOP and MPAK comigrated by native gel electrophoresis, coimmunoprecipitated, and colocalized by immunofluorescence, indicating that MPOP binds to and recruits MPAK to the membrane surface. MPAK, in turn, phosphorylated MFP2 on threonine residues, resulting in incorporation of MFP2 into the cytoskeleton. Beads coated with MPAK assembled a surrounding cloud of MSP filaments when incubated in MPAK-depleted sperm extract, but only when supplemented with detergent-solubilized MPOP. Our results suggest that interactions involving MPOP, MPAK, and MFP2 focus MSP polymerization to the plasma membrane at the leading edge of the cell thereby generating protrusion and minimizing nonproductive filament formation elsewhere.

Yeast formins Bni1 and Bnr1 utilize different modes of cortical interaction during the assembly of actin cables.

The budding yeast formins Bni1 and Bnr1 control the assembly of actin cables. These formins exhibit distinct patterns of localization and polymerize two different populations of cables: Bni1 in the bud and Bnr1 in the mother cell. We generated a functional Bni1-3GFP that improved the visualization of Bni1 in vivo at endogenous levels. Bni1 exists as speckles in the cytoplasm, some of which colocalize on actin cables. These Bni1 speckles display linear, retrograde-directed movements. Loss of polymerized actin or specifically actin cables abolished retrograde movement, and resulted in depletion of Bni1 speckles from the cytoplasm, with enhanced targeting of Bni1 to the bud tip. Mutations that impair the actin assembly activity of Bni1 abolished the movement of Bni1 speckles, even when actin cables were present. In contrast, Bnr1-GFP or 3GFP-Bnr1 did not detectably associate with actin cables and was not observed as cytoplasmic speckles. Finally, fluorescence recovery after photobleaching demonstrated that Bni1 was very dynamic, exchanging between polarized sites and the cytoplasm, whereas Bnr1 was confined to the bud neck and did not exchange with a cytoplasmic pool. In summary, our results indicate that formins can have distinct modes of cortical interaction during actin cable assembly.

Structure of MFP2 and its function in enhancing MSP polymerization in Ascaris sperm amoeboid motility.

The simplicity and specialization of the cell motility machinery of Ascaris sperm provides a powerful system in which to probe the basic molecular mechanism of amoeboid cell motility. Although Ascaris sperm locomotion closely resembles that seen in many other types of crawling cell, movement is generated by modulation of a cytoskeleton based on the major sperm protein (MSP) rather than the actin present in other cell types. The Ascaris motility machinery can be studied conveniently in a cell-free in vitro system based on the movement of plasma membrane vesicles by fibres constructed from bundles of MSP filaments. In addition to ATP, MSP and a plasma membrane protein, reconstitution of MSP motility in this cell-free extract requires cytosolic proteins to orchestrate the site-specific assembly and bundling of MSP filaments that generates locomotion. One of these proteins, MFP2, accelerates the rate of movement in this assay. Here, we describe crystal structures of two isoforms of MFP2 and show that both are constructed from two domains that have the same fold based on a novel, compact beta sheet arrangement. Patterns of conservation observed in a structure-based analysis of MFP2 sequences from different nematode species identified regions that may be putative functional interfaces involved both in interactions between MFP2 domains and also with other components of the sperm motility machinery. Analysis of the growth of fibres in vitro in the presence of added MFP2 indicated that MFP2 increases the rate of locomotion by enhancing the effective rate of MSP filament polymerization. This observation, together with the structural data, suggests that MFP2 may function in a manner analogous to formins in actin-based motility.

Dissection of the Ascaris sperm motility machinery identifies key proteins involved in major sperm protein-based amoeboid locomotion.

Although Ascaris sperm motility closely resembles that seen in many other types of crawling cells, the lamellipodial dynamics that drive movement result from modulation of a cytoskeleton based on the major sperm protein (MSP) rather than actin. The dynamics of the Ascaris sperm cytoskeleton can be studied in a cell-free in vitro system based on the movement of plasma membrane vesicles by fibers constructed from bundles of MSP filaments. In addition to ATP, MSP, and a plasma membrane protein, reconstitution of MSP motility in this cell-free extract requires cytosolic proteins that orchestrate the site-specific assembly and bundling of MSP filaments that generates locomotion. Here, we identify a fraction of cytosol that is comprised of a small number of proteins but contains all of the soluble components required to assemble fibers. We have purified two of these proteins, designated MSP fiber proteins (MFPs) 1 and 2 and demonstrated by immunolabeling that both are located in the MSP cytoskeleton in cells and in fibers. These proteins had reciprocal effects on fiber assembly in vitro: MFP1 decreased the rate of fiber growth, whereas MFP2 increased the growth rate.