Recommendations for accelerating open preprint peer review to improve the culture of science.

Peer review is an important part of the scientific process, but traditional peer review at journals is coming under increased scrutiny for its inefficiency and lack of transparency. As preprints become more widely used and accepted, they raise the possibility of rethinking the peer-review process. Preprints are enabling new forms of peer review that have the potential to be more thorough, inclusive, and collegial than traditional journal peer review, and to thus fundamentally shift the culture of peer review toward constructive collaboration. In this Consensus View, we make a call to action to stakeholders in the community to accelerate the growing momentum of preprint sharing and provide recommendations to empower researchers to provide open and constructive peer review for preprints.

3D Ultrastructural Visualization of Mitosis Fidelity in Human Cells Using Serial Block Face Scanning Electron Microscopy (SBF-SEM).

Errors in chromosome segregation during mitosis lead to chromosome instability, resulting in an unbalanced number of chromosomes in the daughter cells. Light microscopy has been used extensively to study chromosome missegregation by visualizing errors of the mitotic spindle. However, less attention has been paid to understanding spindle function in the broader context of intracellular structures and organelles during mitosis. Here, we outline a protocol to visualize chromosomes and endomembranes in mitosis, combining light microscopy and 3D volume electron microscopy, serial block-face scanning electron microscopy (SBF-SEM). SBF-SEM provides high-resolution imaging of large volumes and subcellular structures, followed by image analysis and 3D reconstruction. This protocol allows scientists to visualize the whole subcellular context of the spindle during mitosis.

Genetically encoded imaging tools for investigating cell dynamics at a glance.

The biology of a cell is the sum of many highly dynamic processes, each orchestrated by a plethora of proteins and other molecules. Microscopy is an invaluable approach to spatially and temporally dissect the molecular details of these processes. Hundreds of genetically encoded imaging tools have been developed that allow cell scientists to determine the function of a protein of interest in the context of these dynamic processes. Broadly, these tools fall into three strategies: observation, inhibition and activation. Using examples for each strategy, in this Cell Science at a Glance and the accompanying poster, we provide a guide to using these tools to dissect protein function in a given cellular process. Our focus here is on tools that allow rapid modification of proteins of interest and how observing the resulting changes in cell states is key to unlocking dynamic cell processes. The aim is to inspire the reader's next set of imaging experiments.

Recruitment of clathrin to intracellular membranes is sufficient for vesicle formation.

The formation of a clathrin-coated vesicle (CCV) is a major membrane remodeling process that is crucial for membrane traffic in cells. Besides clathrin, these vesicles contain at least 100 different proteins although it is unclear how many are essential for the formation of the vesicle. Here, we show that intracellular clathrin-coated formation can be induced in living cells using minimal machinery and that it can be achieved on various membranes, including the mitochondrial outer membrane. Chemical heterodimerization was used to inducibly attach a clathrin-binding fragment 'hook' to an 'anchor' protein targeted to a specific membrane. Endogenous clathrin assembled to form coated pits on the mitochondria, termed MitoPits, within seconds of induction. MitoPits are double-membraned invaginations that form preferentially on high curvature regions of the mitochondrion. Upon induction, all stages of CCV formation - initiation, invagination, and even fission - were faithfully reconstituted. We found no evidence for the functional involvement of accessory proteins in this process. In addition, fission of MitoPit-derived vesicles was independent of known scission factors including dynamins and dynamin-related protein 1 (Drp1), suggesting that the clathrin cage generates sufficient force to bud intracellular vesicles. Our results suggest that, following its recruitment, clathrin is sufficient for intracellular CCV formation.

Integrating intracellular nanovesicles into integrin trafficking pathways and beyond.

Membrane traffic controls the movement of proteins and lipids from one cellular compartment to another using a system of transport vesicles. Intracellular nanovesicles (INVs) are a newly described class of transport vesicles. These vesicles are small, carry diverse cargo, and are involved in multiple trafficking steps including anterograde traffic and endosomal recycling. An example of a biological process that they control is cell migration and invasion, due to their role in integrin recycling. In this review, we describe what is known so far about these vesicles. We discuss how INVs may integrate into established membrane trafficking pathways using integrin recycling as an example. We speculate where in the cell INVs have the potential to operate and we identify key questions for future investigation.

Endomembranes promote chromosome missegregation by ensheathing misaligned chromosomes.

Errors in mitosis that cause chromosome missegregation lead to aneuploidy and micronucleus formation, which are associated with cancer. Accurate segregation requires the alignment of all chromosomes by the mitotic spindle at the metaphase plate, and any misalignment must be corrected before anaphase is triggered. The spindle is situated in a membrane-free "exclusion zone"; beyond this zone, endomembranes (mainly endoplasmic reticulum) are densely packed. We investigated what happens to misaligned chromosomes localized beyond the exclusion zone. Here we show that such chromosomes become ensheathed in multiple layers of endomembranes. Chromosome ensheathing delays mitosis and increases the frequency of chromosome missegregation and micronucleus formation. We use an induced organelle relocalization strategy in live cells to show that clearance of endomembranes allows for the rescue of chromosomes that were destined for missegregation. Our findings indicate that endomembranes promote the missegregation of misaligned chromosomes that are outside the exclusion zone and therefore constitute a risk factor for aneuploidy.

Multi-modal adaptor-clathrin contacts drive coated vesicle assembly.

Clathrin-coated pits are formed by the recognition of membrane and cargo by the AP2 complex and the subsequent recruitment of clathrin triskelia. A role for AP2 in coated-pit assembly beyond initial clathrin recruitment has not been explored. Clathrin binds the ß2 subunit of AP2, and several binding sites have been identified, but our structural knowledge of these interactions is incomplete and their functional importance during endocytosis is unclear. Here, we analysed the cryo-EM structure of clathrin cages assembled in the presence of ß2 hinge-appendage (ß2HA). We find that the ß2-appendage binds in at least two positions in the cage, demonstrating that multi-modal binding is a fundamental property of clathrin-AP2 interactions. In one position, ß2-appendage cross-links two adjacent terminal domains from different triskelia. Functional analysis of ß2HA-clathrin interactions reveals that endocytosis requires two clathrin interaction sites: a clathrin-box motif on the hinge and the "sandwich site" on the appendage. We propose that ß2-appendage binding to more than one triskelion is a key feature of the system and likely explains why assembly is driven by AP2.

Intracellular nanovesicles mediate α5ß1 integrin trafficking during cell migration.

Membrane traffic is an important regulator of cell migration through the endocytosis and recycling of cell surface receptors such as integrin heterodimers. Intracellular nanovesicles (INVs) are transport vesicles that are involved in multiple membrane trafficking steps, including the recycling pathway. The only known marker for INVs is tumor protein D54 (TPD54/TPD52L2), a member of the TPD52-like protein family. Overexpression of TPD52-like family proteins in cancer has been linked to poor prognosis and an aggressive metastatic phenotype, which suggests cell migration may be altered under these conditions. Here, we show that TPD54 directly binds membrane and associates with INVs via a conserved positively charged motif in its C terminus. We describe how other TPD52-like proteins are also associated with INVs, and we document the Rab GTPase complement of all INVs. Depletion of TPD52-like proteins inhibits cell migration and invasion, while their overexpression boosts motility. We show that inhibition of migration is likely due to altered recycling of α5ß1 integrins in INVs.

Defining endogenous TACC3-chTOG-clathrin-GTSE1 interactions at the mitotic spindle using induced relocalization.

A multiprotein complex containing TACC3, clathrin and other proteins has been implicated in mitotic spindle stability. To disrupt this complex in an anti-cancer context, we need to understand its composition and how it interacts with microtubules. Induced relocalization of proteins in cells is a powerful way to analyze protein-protein interactions and, additionally, monitor where and when these interactions occur. We used CRISPR/Cas9 gene editing to add tandem FKBP-GFP tags to each complex member. The relocalization of endogenous tagged protein from the mitotic spindle to mitochondria and assessment of the effect on other proteins allowed us to establish that TACC3 and clathrin are core complex members and that chTOG (also known as CKAP5) and GTSE1 are ancillary to the complex, binding respectively to TACC3 and clathrin, but not each other. We also show that PIK3C2A, a clathrin-binding protein that was proposed to stabilize the TACC3-chTOG-clathrin-GTSE1 complex during mitosis, is not a member of the complex. This work establishes that targeting the TACC3-clathrin interface or their microtubule-binding sites are the two strategies most likely to disrupt spindle stability mediated by this multiprotein complex.

Tumor protein D54 defines a new class of intracellular transport vesicles.

Transport of proteins and lipids from one membrane compartment to another is via intracellular vesicles. We investigated the function of tumor protein D54 (TPD54/TPD52L2) and found that TPD54 was involved in multiple membrane trafficking pathways: anterograde traffic, recycling, and Golgi integrity. To understand how TPD54 controls these diverse functions, we used an inducible method to reroute TPD54 to mitochondria. Surprisingly, this manipulation resulted in the capture of many small vesicles (30 nm diameter) at the mitochondrial surface. Super-resolution imaging confirmed the presence of similarly sized TPD54-positive structures under normal conditions. It appears that TPD54 defines a new class of transport vesicle, which we term intracellular nanovesicles (INVs). INVs meet three criteria for functionality. They contain specific cargo, they have certain R-SNAREs for fusion, and they are endowed with a variety of Rab GTPases (16 out of 43 tested). The molecular heterogeneity of INVs and the diverse functions of TPD54 suggest that INVs have various membrane origins and a number of destinations. We propose that INVs are a generic class of transport vesicle that transfer cargo between these varied locations.

Unintended perturbation of protein function using GFP nanobodies in human cells.

Tagging a protein of interest with GFP using genome editing is a popular approach to study protein function in cell and developmental biology. To avoid re-engineering cell lines or organisms in order to introduce additional tags, functionalized nanobodies that bind GFP can be used to extend the functionality of the GFP tag. We developed functionalized nanobodies, which we termed 'dongles', that could add, for example, an FKBP tag to a GFP-tagged protein of interest, enabling knocksideways experiments in GFP knock-in cell lines. The power of knocksideways is that it allows investigators to rapidly switch the protein from an active to an inactive state. We show that dongles allow for effective knocksideways of GFP-tagged proteins in genome-edited human cells. However, we discovered that nanobody binding to dynamin-2-GFP caused inhibition of dynamin function prior to knocksideways. The function of GFP-tagged tumor protein D54 (TPD54, also known as TPD52L2) in anterograde traffic was also perturbed by dongles. While these issues potentially limit the application of dongles, we discuss strategies for their deployment as cell biological tools.This article has an associated First Person interview with the first author of the paper.

The NAE Pathway: Autobahn to the Nucleus for Cell Surface Receptors.

Various growth factors and full-length cell surface receptors such as EGFR are translocated from the cell surface to the nucleoplasm, baffling cell biologists to the mechanisms and functions of this process. Elevated levels of nuclear EGFR correlate with poor prognosis in various cancers. In recent years, nuclear EGFR has been implicated in regulating gene transcription, cell proliferation and DNA damage repair. Different models have been proposed to explain how the receptors are transported into the nucleus. However, a clear consensus has yet to be reached. Recently, we described the nuclear envelope associated endosomes (NAE) pathway, which delivers EGFR from the cell surface to the nucleus. This pathway involves transport, docking and fusion of NAEs with the outer membrane of the nuclear envelope. EGFR is then presumed to be transported through the nuclear pore complex, extracted from membranes and solubilised. The SUN1/2 nuclear envelope proteins, Importin-beta, nuclear pore complex proteins and the Sec61 translocon have been implicated in the process. While this framework can explain the cell surface to nucleus traffic of EGFR and other cell surface receptors, it raises several questions that we consider in this review, together with implications for health and disease.

Imaging "Hot-Wired" Clathrin-Mediated Endocytosis.

Clathrin-mediated endocytosis (CME) occurs continuously at the plasma membrane of eukaryotic cells. However, when a vesicle forms and what cargo it contains are unpredictable. We recently developed a system to trigger CME on-demand. This means that we can control when endocytosis is triggered and the design means that the cargo that is internalized is predetermined. The method is called hot-wired CME because several steps and proteins are bypassed in our system. In this chapter, we describe in detail how to use the hot-wiring system to trigger endocytosis in human cell lines and how to image the vesicles that form using microscopy and finally, how to analyze those images.

FerriTag is a new genetically-encoded inducible tag for correlative light-electron microscopy.

A current challenge is to develop tags to precisely visualize proteins in cells by light and electron microscopy. Here, we introduce FerriTag, a genetically-encoded chemically-inducible tag for correlative light-electron microscopy. FerriTag is a fluorescent recombinant electron-dense ferritin particle that can be attached to a protein-of-interest using rapamycin-induced heterodimerization. We demonstrate the utility of FerriTag for correlative light-electron microscopy by labeling proteins associated with various intracellular structures including mitochondria, plasma membrane, and clathrin-coated pits and vesicles. FerriTagging has a good signal-to-noise ratio and a labeling resolution of approximately 10 nm. We demonstrate how FerriTagging allows nanoscale mapping of protein location relative to a subcellular structure, and use it to detail the distribution and conformation of huntingtin-interacting protein 1 related (HIP1R) in and around clathrin-coated pits.

Correlating light microscopy with serial block face scanning electron microscopy to study mitotic spindle architecture.

The mitotic spindle is a complex structure that coordinates the accurate segregation of chromosomes during cell division. To understand how the mitotic spindle operates at the molecular level, high resolution imaging is needed. Serial block face-scanning electron microscopy (SBF-SEM) is a technique that can be used to visualize the ultrastructure of entire cells, including components of the mitotic spindle such as microtubules, kinetochores, centrosomes, and chromosomes. Although transmission electron microscopy (TEM) has higher resolution, the reconstruction of large volumes using TEM and tomography is labor intensive, whereas SBF-SEM takes only days to process, image, and segment samples. SBF-SEM fills the resolution gap between light microscopy (LM) and TEM. When combined with LM, SBF-SEM provides a platform where dynamic cellular events can be selected and imaged at high resolution. Here we outline methods to use correlation and SBF-SEM to study mitotic spindle architecture in 3D with high resolution.

Mitotic spindle association of TACC3 requires Aurora-A-dependent stabilization of a cryptic α-helix.

Aurora-A regulates the recruitment of TACC3 to the mitotic spindle through a phospho-dependent interaction with clathrin heavy chain (CHC). Here, we describe the structural basis of these interactions, mediated by three motifs in a disordered region of TACC3. A hydrophobic docking motif binds to a previously uncharacterized pocket on Aurora-A that is blocked in most kinases. Abrogation of the docking motif causes a delay in late mitosis, consistent with the cellular distribution of Aurora-A complexes. Phosphorylation of Ser558 engages a conformational switch in a second motif from a disordered state, needed to bind the kinase active site, into a helical conformation. The helix extends into a third, adjacent motif that is recognized by a helical-repeat region of CHC, not a recognized phospho-reader domain. This potentially widespread mechanism of phospho-recognition provides greater flexibility to tune the molecular details of the interaction than canonical recognition motifs that are dominated by phosphate binding.

FGFR3-TACC3 cancer gene fusions cause mitotic defects by removal of endogenous TACC3 from the mitotic spindle.

Fibroblast growth factor receptor 3-transforming acidic coiled-coil containing protein 3 (FGFR3-TACC3; FT3) is a gene fusion resulting from rearrangement of chromosome 4 that has been identified in many cancers including those of the urinary bladder. Altered FGFR3 signalling in FT3-positive cells is thought to contribute to cancer progression. However, potential changes in TACC3 function in these cells have not been explored. TACC3 is a mitotic spindle protein required for accurate chromosome segregation. Errors in segregation lead to aneuploidy, which can contribute to cancer progression. Here we show that FT3-positive bladder cancer cells have lower levels of endogenous TACC3 on the mitotic spindle, and that this is sufficient to cause mitotic defects. FT3 is not localized to the mitotic spindle, and by virtue of its TACC domain, recruits endogenous TACC3 away from the spindle. Knockdown of the fusion gene or low-level overexpression of TACC3 partially rescues the chromosome segregation defects in FT3-positive bladder cancer cells. This function of FT3 is specific to TACC3 as inhibition of FGFR3 signalling does not rescue the TACC3 level on the spindle in these cancer cells. Models of FT3-mediated carcinogenesis should, therefore, include altered mitotic functions of TACC3 as well as altered FGFR3 signalling.

New tools for "hot-wiring" clathrin-mediated endocytosis with temporal and spatial precision.

Clathrin-mediated endocytosis (CME) is the major route of receptor internalization at the plasma membrane. Analysis of constitutive CME is difficult because the initiation of endocytic events is unpredictable. When and where a clathrin-coated pit will form and what cargo it will contain are difficult to foresee. Here we describe a series of genetically encoded reporters that allow the initiation of CME on demand. A clathrin-binding protein fragment ("hook") is inducibly attached to an "anchor" protein at the plasma membrane, which triggers the formation of new clathrin-coated vesicles. Our design incorporates temporal and spatial control by the use of chemical and optogenetic methods for inducing hook-anchor attachment. Moreover, the cargo is defined. Because several steps in vesicle creation are bypassed, we term it "hot-wiring." We use hot-wired endocytosis to describe the functional interactions between clathrin and AP2. Two distinct sites on the ß2 subunit, one on the hinge and the other on the appendage, are necessary and sufficient for functional clathrin engagement.

Congressing kinetochores progressively load Ska complexes to prevent force-dependent detachment.

Kinetochores mediate chromosome congression by either sliding along the lattice of spindle microtubules or forming end-on attachments to their depolymerizing plus-ends. By following the fates of individual kinetochores as they congress in live cells, we reveal that the Ska complex is required for a distinct substep of the depolymerization-coupled pulling mechanism. Ska depletion increases the frequency of naturally occurring, force-dependent P kinetochore detachment events, while being dispensable for the initial biorientation and movement of chromosomes. In unperturbed cells, these release events are followed by reattachment and successful congression, whereas in Ska-depleted cells, detached kinetochores remain in a futile reattachment/detachment cycle that prevents congression. We further find that Ska is progressively loaded onto bioriented kinetochore pairs as they congress. We thus propose a model in which kinetochores mature through Ska complex recruitment and that this is required for improved load-bearing capacity and silencing of the spindle assembly checkpoint.

Microtubule organization within mitotic spindles revealed by serial block face scanning electron microscopy and image analysis.

Serial block face scanning electron microscopy (SBF-SEM) is a powerful method to analyze cells in 3D. Here, working at the resolution limit of the method, we describe a correlative light-SBF-SEM workflow to resolve microtubules of the mitotic spindle in human cells. We present four examples of uses for this workflow that are not practical by light microscopy and/or transmission electron microscopy. First, distinguishing closely associated microtubules within K-fibers; second, resolving bridging fibers in the mitotic spindle; third, visualizing membranes in mitotic cells, relative to the spindle apparatus; and fourth, volumetric analysis of kinetochores. Our workflow also includes new computational tools for exploring the spatial arrangement of microtubules within the mitotic spindle. We use these tools to show that microtubule order in mitotic spindles is sensitive to the level of TACC3 on the spindle.