Understanding the spatiotemporal dependencies between different protein species, as observed through fluorescent confocal laser microscopy, is crucial for gaining insight into their biological functions. We introduce an estimator of their bulk movement patterns between time points on a space, Ψ, using the earth mover's distance. We propose a test statistic that combines these bulk movement patterns over a partition, Ψw, of Ψ into w subregions and compares them between two samples. At the core of our approach lies a novel null hypothesis framework, consisting of statements regarding between- and within-sample independence of bulk movement patterns, alongside a statement of distributional invariance under the operation of a geometrically defined subgroup of permutations acting on Ψw. This framework yields a geometrically informed permutation (GIP) test designed to quantify the significance of dependencies between bulk movement patterns. We validate the approach using synthetic data spanning a range of independent and dependent scenarios with varying geometrical properties. Finally, we apply the GIP test to experiments involving the microtubule-associated proteins EB3 and TACC3, obtaining evidence that reinforces previous biological findings on the colocalisation of these proteins. More broadly, our proposed methodology is applicable to a wide range of spatiotemporal molecular data problems in which geometric structure is fundamental, particularly when the surrounding cellular or tissue environment informs the dynamics.

Mitotic spindle stability is enhanced through microtubule crosslinking by a complex formed when an α helix in transforming acidic coiled-coil 3 (TACC3) binds to the helical repeats of the heavy chain of clathrin (CHC). Here, we show that the phosphorylation of TACC3 at S558 modulates the interaction by overcoming the electrostatic repulsion between K507 of CHC and basic residues in TACC3. Leveraging this insight, we optimized the sequence using peptide arrays to develop a hydrocarbon-stapled peptide (SP TACC3) that binds CHC with over 400-fold higher affinity than the native sequence, disrupting the interaction. The crystal structure of the SP TACC3-CHC complex reveals the contribution of additional polar and hydrophobic contacts to the enhanced interaction. SP TACC3 penetrates cells and displaces TACC3 from the mitotic spindle, causing a delay in mitotic progression in two out of three cancer cell lines. This work showcases a strategy for targeting the TACC3-CHC interaction with hydrocarbon-stapled peptides in a cellular context for potential cancer therapies.

The discovery of clathrin was a foundational event in membrane traffic research. Its identification as the major protein component of the coat that surrounds endocytic vesicles kickstarted the biochemical and molecular characterization of endocytosis. During this explosive period of cell biology, there was a parallel storyline involving clathrin which developed more slowly. It emerged that clathrin has an alternative function during mitosis, and that this function is unrelated to its membrane trafficking role. Clathrin forms part of a multiprotein complex that stabilizes microtubules of the mitotic spindle during the chromosome segregation events that occur during cell division. Due to this dual functionality, clathrin is sometimes referred to as a "moonlighting" protein. In this Perspective, we will take a look at this secret life of clathrin and examine how it carries out its mitotic function.

The complex formed by transforming acidic coiled coil 3 (TACC3) and clathrin heavy chain (CHC) enhances mitotic spindle stability and strength by cross-linking microtubules. The interaction is dependent on phosphorylation of TACC3 at S558 by Aurora-A. Previously, we elucidated the structural basis of the TACC3/CHC interaction, which is driven by hydrophobic residues on both proteins and the formation of an α-helix in TACC3 that docks into the helical repeats of CHC. Here we find that this phosphorylation event plays an unusual role in the protein-protein interaction; rather than direct bond formation, the phosphorylated residue acts by overcoming an inherent electrostatic repulsion between K507 of CHC and basic residues in TACC3. Leveraging this insight, we optimized the sequence using peptide arrays to develop a hydrocarbon-stapled peptide (SP TACC3) that binds CHC with over a hundred-fold higher affinity than the parental TACC3 peptide, effectively disrupting the native interaction. The crystal structure of the SP TACC3/CHC complex reveals the basis for the enhanced interaction and highlights the contribution of additional polar and hydrophobic interactions. SP TACC3 efficiently penetrates cells and displaces TACC3 from the mitotic spindle, causing a delay in mitotic progression in two out of three cancer cell lines. This work showcases the novel application of hydrocarbon-stapled peptides to disrupt the TACC3/CHC protein-protein interaction in a cellular context, highlighting the potential of targeting this interface for future cancer therapies.

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The mitotic spindle is a bipolar array of microtubules, radiating from the poles which each contain a centrosome, embedded in pericentriolar material. Two proteins, ch-TOG and TACC3, have multiple functions at the mitotic spindle due to operating either alone, together, or in complex with other proteins. To distinguish these activities, we need new molecular tools to dissect their function. Here, we present the structure of the α-helical bundle domain of ch-TOG that mediates its interaction with TACC3 and a structural model describing the interaction, supported by biophysical and biochemical data. We have isolated Affimer tools to precisely target the ch-TOG-binding site on TACC3 in live cells, which displace ch-TOG without affecting the spindle localization of other protein complex components. Inhibition of the TACC3–ch-TOG interaction led unexpectedly to fragmentation of the pericentriolar material in metaphase cells and delayed mitotic progression, uncovering a novel role of TACC3–ch-TOG in maintaining pericentriolar material integrity during mitosis to ensure timely cell division.

Cells are filled with thousands of vesicles, which mediate protein transport and ensure homeostasis of the endomembrane system. Distinguishing these vesicles functionally and molecularly represents a major challenge. Intracellular nanovesicles (INVs) are a large class of transport vesicles that likely comprise multiple subtypes. Here, we define the INV proteome and find that it is molecularly heterogeneous and enriched for transmembrane cargo molecules, including integrins, transporters and ATG9A, a lipid scramblase associated with autophagy. ATG9A is known to reside in 'ATG9A vesicles' - small vesicles that contribute to autophagosome formation. Here, using in-cell vesicle capture assays, we found that ATG9A, as well as other ATG9A vesicle cargoes, are in INVs. Quantitative analysis showed that virtually all ATG9A vesicles are INVs, but that only ∼20% of INVs are ATG9A vesicles, suggesting that ATG9A vesicles are in fact a subtype of INV, which we term ATG9A-flavor INVs. Finally, we show that perturbing ATG9A-flavor INVs impairs the autophagy response induced by starvation.

The molecular and functional characterization of the thousands of uncoated intracellular transport vesicles inside cells is a major challenge. Intracellular nanovesicles (INVs) are a large and molecularly heterogenous family of uncoated transport vesicles, which are comprised of multiple subtypes. As a step to characterizing these subtypes, we recently published the first INV proteome and were intrigued by the enrichment of ATG9A in it. ATG9A is the only conserved transmembrane protein with a core function in macroautophagy/autophagy, and it is found on small, uncoated vesicles, termed “ATG9A-positive vesicles”. We therefore, set out to disambiguate the relationship between these two types of vesicular carriers in cells. We showed that ATG9A-containing vesicles, rather than being a distinct vesicle class, represent one subset of the INV family. We also demonstrated that this relationship is functionally important and that perturbing INV-mediated trafficking impeded starvation-induced autophagy. Here, we briefly introduce INVs, summarize the evidence supporting our definition of ATG9A-flavor INVs and present our outlook on why we hope that this classification will help to consolidate efforts to understand the functions of these vesicles in autophagy and beyond.

Membrane contact sites (MCSs) are areas of close proximity between organelles that allow the exchange of material, among other roles. The endoplasmic reticulum (ER) has MCSs with a variety of organelles in the cell. MCSs are dynamic, responding to changes in cell state, and are, therefore, best visualized through inducible labeling methods. However, existing methods typically distort ER-MCSs, by expanding contacts or creating artificial ones. Here, we describe a new method for inducible labeling of ER-MCSs using the Lamin B receptor (LBR) and a generic anchor protein on the partner organelle. Termed LaBeRling, this versatile, one-to-many approach allows labeling of different types of ER-MCSs (mitochondria, plasma membrane, lysosomes, early endosomes, lipid droplets, and Golgi), on-demand, in interphase or mitotic human cells. LaBeRling is nondisruptive and does not change ER-MCSs in terms of the contact number, extent or distance measured; as determined by light microscopy or a deep-learning volume electron microscopy approach. We applied this method to study the changes in ER-MCSs during mitosis and to label novel ER-Golgi contact sites at different mitotic stages in live cells.

Integrins are heterodimeric cell surface receptors that govern cell-cell interactions, which in turn can influence multiscale processes: cell migration, extracellular matrix remodeling and tissue formation. These processes occur over timescales which range from milliseconds to days. While various strategies exist to study integrin function across biological scales from cell to tissue, they are often chronic and fail to target specific cell-cell interactions acutely. We engineered cells to rapidly alter cell behavior by downregulating the surface population of α5β1 integrins through hot-wired clathrin-mediated endocytosis. This method allows for inducible, specific internalization of α5β1 integrins, achieving acute downregulation across various cell lines in 5-30 minutes. We show that induced internalization of α5β1 decreases the cell area, causes uptake of extracellular fibronectin, and decreases the rate of tumor spheroid compaction. This targeted control of multiscale processes by rapid downregulation of this important class of cell surface receptors demonstrates that hot-wired endocytosis is a useful tool to acutely modulate cell biology. ### Competing Interest Statement The authors have declared no competing interest.

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.

Clathrins are self-assembling cytoplasmic proteins that serve to mediate membrane trafficking. At intracellular membranes, individual clathrin subunits surround the invaginating membrane to form a protein coat that assists in cargo capture and vesicle formation. The protein coat is a polyhedral, multimeric assembly of clathrin triskelia (3-legged structures partly composed of 3 clathrin heavy chains (CHC)). There are two forms of CHC in vertebrates CHC17 and CHC22 which have distinct cellular functions. CHC17 is implicated in receptor-mediated endocytosis at the plasma membrane and organelle biogenesis at the trans-Golgi network. During clathrin-mediated endocytosis, CHC17 cannot recognise membrane or cargo and so an adaptor protein binds the membrane, selects the cargo, and associates with clathrin leading to pit formation. Several adaptor proteins have clathrin binding sites and colocalize with clathrin structures in cells. Our structural knowledge of these adaptor-clathrin interactions, and their functional importance, is unclear. We analysed the cryo-EM structure of CHC17 cages assembled in the presence of the clathrin-binding subunit (β2-appendage) of assembly polypeptide-2 (AP2) (the adaptor protein that is thought to primarily initiate clathrin recruitment). We found that the β2-appendage binds in at least two positions in the cage. We propose that β2-appendage binding to more than one triskelion is a key feature of the system and likely explains why clathrin assembly is driven by AP2. These data then led us to ask: is multi-modal binding a fundamental property of clathrin-adaptor interactions? CHC22 acts to sequester the GLUT4 glucose transporter in an insulin-responsive compartment - a behaviour critical to controlling blood sugar levels. Understanding how clathrin self-assembles into basket-like structures to facilitate such cellular functions will significantly advance our understanding of clathrin biology. To this end, we endeavor to map the molecular structure of CHC22.

Preprints enable new forms of peer review that have the potential to be more thorough, inclusive, and collegial. In December 2022, 80 researchers and representatives of funders, institutions, preprint servers, journals, indexers, and review services were invited to gather online and at the Janelia Research Campus for a workshop on Recognizing Preprint Peer Review. Sponsored by HHMI, ASAPbio, and EMBO, this meeting aimed to catalyze community consensus and support for preprint peer review and to create model funder, institutional, and journal policies that recognize both preprints with reviews, and reviews of preprints. Here, we make a call to action to stakeholders in the community to help capture the growing momentum of preprint sharing and empower researchers to provide open and constructive peer review for preprints.

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.

During membrane trafficking, a vesicle formed at the donor compartment must travel to the acceptor membrane before fusing. For large carriers, it is established that this transport is motor-driven; however, the mode by which small vesicles, which outnumber larger carriers, are transported is poorly characterized. Here we show that intracellular nanovesicles (INVs), a substantial class of small vesicles, are highly mobile within cells and that this mobility depends almost entirely on passive diffusion (0.1–0.3 µm 2 s −1 ). Using single particle tracking, we describe how other small trafficking vesicles have a similar diffusive mode of transport, that contrasts with the motor-dependent movement of larger endolysosomal carriers. We also demonstrate that a subset of INVs are involved in constitutive secretion and that delivery of cargo to the plasma membrane during exocytosis is decreased when diffusion of INVs is specifically restricted. Our results suggest that passive diffusion is sufficient to explain the majority of small vesicle transport.

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.

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.

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.

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.