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.

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.

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.

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.

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.

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.

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.