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Molecular pathways that intrinsically regulate neuronal maintenance are poorly understood, but rare pathogenic mutations that underlie neurodegenerative disease can offer important insights into the mechanisms that facilitate lifelong neuronal function. Here, we leverage a rat model to demonstrate directly that the TFG p.R106C variant implicated previously in complicated forms of hereditary spastic paraplegia (HSP) underlies progressive spastic paraparesis with accompanying ventriculomegaly and thinning of the corpus callosum, consistent with disease phenotypes identified in adolescent patients. Analyses of primary cortical neurons obtained from CRISPR-Cas9–edited animals reveal a kinetic delay in biosynthetic secretory protein transport from the endoplasmic reticulum (ER), in agreement with prior induced pluripotent stem cell–based studies. Moreover, we identify an unexpected role for TFG in the trafficking of Rab4A-positive recycling endosomes specifically within axons and dendrites. Impaired TFG function compromises the transport of at least a subset of endosomal cargoes, which we show results in down-regulated inhibitory receptor signaling that may contribute to excitation-inhibition imbalances. In contrast, the morphology and trafficking of other organelles, including mitochondria and lysosomes, are unaffected by the TFG p.R106C mutation. Our findings demonstrate a multifaceted role for TFG in secretory and endosomal protein sorting that is unique to cells of the central nervous system and highlight the importance of these pathways to maintenance of corticospinal tract motor neurons.

Protein-protein interactions constitute the molecular foundations of virtually all biological processes. Co-immunoprecipitation (CoIP) experiments are probably the most widely used method to probe both heterotypic and homotypic protein-protein interactions. Recent advances in super-resolution microscopy have revealed that several nuclear proteins such as transcription factors are spatially distributed into local high-concentration clusters in mammalian cells, suggesting that many nuclear proteins self-interact. These observations have further underscored the need for orthogonal biochemical approaches for testing if self-association occurs, and if so, what the mechanisms are. Here, we describe a CoIP protocol specifically optimized to test self-association of endogenously tagged nuclear proteins (self-CoIP), and to evaluate the role of nucleic acids in such self-interaction. This protocol has proven reliable and robust in our hands, and it can be used to test both homotypic and heterotypic (CoIP) protein-protein interactions.

Coat proteins play multiple roles in the life cycle of a membrane-bound transport intermediate, functioning in lipid bilayer remodeling, cargo selection and targeting to an acceptor compartment. The Coat Protein complex II (COPII) coat is known to act in each of these capacities, but recent work highlights the necessity for numerous accessory factors at all stages of transport carrier existence. Here, we review recent findings that highlight the roles of COPII and its regulators in the biogenesis of tubular COPII-coated carriers in mammalian cells that enable cargo transport between the endoplasmic reticulum and ER-Golgi intermediate compartments, the first step in a series of trafficking events that ultimately allows for the distribution of biosynthetic secretory cargoes throughout the entire endomembrane system.

Mammalian genomes are folded into topologically associating domains (TADs), consisting of chromatin loops anchored by CTCF and cohesin. Some loops are cell-type specific. Here we asked whether CTCF loops are established by a universal or locus-specific mechanism. Investigating the molecular determinants of CTCF clustering, we found that CTCF self-association in vitro is RNase sensitive and that an internal RNA-binding region (RBR(i)) mediates CTCF clustering and RNA interaction in vivo. Strikingly, deleting the RBR(i) impairs about half of all chromatin loops in mESCs and causes deregulation of gene expression. Disrupted loop formation correlates with diminished clustering and chromatin binding of RBR(i) mutant CTCF, which in turn results in a failure to halt cohesion-mediated extrusion. Thus, CTCF loops fall into at least two classes: RBR(i)-independent and RBR(i-)dependent loops. We speculate that evidence for RBR(i)-dependent loops may provide a molecular mechanism for establishing cell-specific CTCF loops, potentially regulated by RNA(s) or other RBR(i)-interacting partners.

Achieving a quantitative and predictive understanding of 3D genome architecture remains a major challenge, as it requires quantitative measurements of the key proteins involved. Here, we report the quantification of CTCF and cohesin, two causal regulators of topologically associating domains (TADs) in mammalian cells. Extending our previous imaging studies (Hansen et al., 2017), we estimate bounds on the density of putatively DNA loop-extruding cohesin complexes and CTCF binding site occupancy. Furthermore, co-immunoprecipitation studies of an endogenously tagged subunit (Rad21) suggest the presence of cohesin dimers and/or oligomers. Finally, based on our cell lines with accurately measured protein abundances, we report a method to conveniently determine the number of molecules of any Halo-tagged protein in the cell. We anticipate that our results and the established tool for measuring cellular protein abundances will advance a more quantitative understanding of 3D genome organization, and facilitate protein quantification, key to comprehend diverse biological processes.

Summary Length-dependent axonopathy of the corticospinal tract causes lower limb spasticity and is characteristic of several neurological disorders, including hereditary spastic paraplegia (HSP) and amyotrophic lateral sclerosis. Mutations in Trk-fused gene (TFG) have been implicated in both diseases, but the pathomechanisms by which these alterations cause neuropathy remain unclear. Here, we biochemically and genetically define the impact of a mutation within the TFG coiled-coil domain, which underlies early-onset forms of HSP. We find that the TFG (p.R106C) mutation alters compaction of TFG ring complexes, which play a critical role in the export of cargoes from the endoplasmic reticulum (ER). Using CRISPR-mediated genome editing, we engineered human stem cells that express the mutant form of TFG at endogenous levels and identified specific defects in secretion from the ER and axon fasciculation following neuronal differentiation. Together, our data highlight a key role for TFG-mediated protein transport in the pathogenesis of HSP.

Folding of mammalian genomes into spatial domains is critical for gene regulation. The insulator protein CTCF and cohesin control domain location by folding domains into loop structures, which are widely thought to be stable. Combining genomic and biochemical approaches we show that CTCF and cohesin co-occupy the same sites and physically interact as a biochemically stable complex. However, using single-molecule imaging we find that CTCF binds chromatin much more dynamically than cohesin (~1–2 min vs. ~22 min residence time). Moreover, after unbinding, CTCF quickly rebinds another cognate site unlike cohesin for which the search process is long (~1 min vs. ~33 min). Thus, CTCF and cohesin form a rapidly exchanging 'dynamic complex' rather than a typical stable complex. Since CTCF and cohesin are required for loop domain formation, our results suggest that chromatin loops are dynamic and frequently break and reform throughout the cell cycle. DOI: http://dx.doi.org/10.7554/eLife.25776.001, eLife digest A human cell contains about 2 meters of DNA tightly packed in a compartment called the nucleus. Within the space inside the nucleus, different parts of the DNA fold into distinct bundles known as domains. These domains are important for organising the genome and are crucial for regulating gene expression, by stimulating specific DNA segments to activate certain genes. Previous research has shown that DNA segments within the same domain frequently interact, whereas DNA segments in different domains rarely do. The domains are often folded into loops that are held together by a ring-shaped protein complex called cohesin, while another protein called CTCF positions cohesin and thereby sets the boundaries between the domains. Some mutations are known to disrupt these boundaries, which allows certain DNA segments to activate the wrong genes. This can lead to cancer or cause defects when embryos are developing. However, we do not currently understand how these domains are formed or maintained. In particular, it was unclear whether these loop domains are stable or dynamic structures. Hansen et al. addressed these questions in embryonic stem cells from mice and human cancer cells. It was found that cohesin and CTCF form a complex that binds to the DNA and likely holds the loops together. In further experiments, single molecules of cohesin and CTCF were tracked inside cells using super-resolution microscopy. The results showed that CTCF and cohesin bind to DNA with different dynamics: CTCF binds the DNA for about a minute, whereas cohesin binds the DNA for about 20–25 minutes. Once CTCF detaches from DNA, it quickly rebinds DNA at another site, but cohesin takes much longer. These observations suggest that rather than remaining static, chromatin domains are held together by a dynamic protein complex, with a molecular composition that exchanges over time. This results suggests that DNA loop domains, which were generally assumed to be very stable anchor points, are in fact highly dynamic structures that frequently fall apart and reform. The next challenge will be to understand how the dynamic nature of these loop domains contribute to gene regulation. This may, one day, enable us to manipulate the domains to correct faulty folding of DNA in cancer and other diseases. DOI: http://dx.doi.org/10.7554/eLife.25776.002