Approximately one-third of global CO fixation occurs in a phase-separated algal organelle called the pyrenoid. The existing data suggest that the pyrenoid forms by the phase separation of the CO-fixing enzyme Rubisco with a linker protein; however, the molecular interactions underlying this phase separation remain unknown. Here we present the structural basis of the interactions between Rubisco and its intrinsically disordered linker protein Essential Pyrenoid Component 1 (EPYC1) in the model alga Chlamydomonas reinhardtii. We find that EPYC1 consists of five evenly spaced Rubisco-binding regions that share sequence similarity. Single-particle cryo-electron microscopy of these regions in complex with Rubisco indicates that each Rubisco holoenzyme has eight binding sites for EPYC1, one on each Rubisco small subunit. Interface mutations disrupt binding, phase separation and pyrenoid formation. Cryo-electron tomography supports a model in which EPYC1 and Rubisco form a codependent multivalent network of specific low-affinity bonds, giving the matrix liquid-like properties. Our results advance the structural and functional understanding of the phase separation underlying the pyrenoid, an organelle that plays a fundamental role in the global carbon cycle.

Approximately one-third of global CO fixation occurs in a phase-separated algal organelle called the pyrenoid. The existing data suggest that the pyrenoid forms by the phase separation of the CO-fixing enzyme Rubisco with a linker protein; however, the molecular interactions underlying this phase separation remain unknown. Here we present the structural basis of the interactions between Rubisco and its intrinsically disordered linker protein Essential Pyrenoid Component 1 (EPYC1) in the model alga Chlamydomonas reinhardtii. We find that EPYC1 consists of five evenly spaced Rubisco-binding regions that share sequence similarity. Single-particle cryo-electron microscopy of these regions in complex with Rubisco indicates that each Rubisco holoenzyme has eight binding sites for EPYC1, one on each Rubisco small subunit. Interface mutations disrupt binding, phase separation and pyrenoid formation. Cryo-electron tomography supports a model in which EPYC1 and Rubisco form a codependent multivalent network of specific low-affinity bonds, giving the matrix liquid-like properties. Our results advance the structural and functional understanding of the phase separation underlying the pyrenoid, an organelle that plays a fundamental role in the global carbon cycle.

In this paper, we provide a historical account of the contribution of a single line of research to our current understanding of the structure of cis-regulatory regions and the genetic basis for morphological evolution. We revisit the experiments that shed light on the evolution of larval cuticular patterns within the genus Drosophila and the evolution and structure of the shavenbaby gene. We describe the experiments that led to the discovery that multiple genetic changes in the cis-regulatory region of shavenbaby caused the loss of dorsal cuticular hairs (quaternary trichomes) in first instar larvae of Drosophila sechellia. We also discuss the experiments that showed that the convergent loss of quaternary trichomes in D. sechellia and Drosophila ezoana was generated by parallel genetic changes in orthologous enhancers of shavenbaby. We discuss the observation that multiple shavenbaby enhancers drive overlapping patterns of expression in the embryo and that these apparently redundant enhancers ensure robust shavenbaby expression and trichome morphogenesis under stressful conditions. All together, these data, collected over 13 years, provide a fundamental case study in the fields of gene regulation and morphological evolution, and highlight the importance of prolonged, detailed studies of single genes.

The ubiquitous members of the aquaporin (AQP) family form transmembrane pores that are either exclusive for water (aquaporins) or are also permeable for other small neutral solutes such as glycerol (aquaglyceroporins). The purpose of this review is to provide an overview of our current knowledge of AQP structures and to describe the structural features that define the function of these membrane pores. The review will discuss the mechanisms governing water conduction, proton exclusion and substrate specificity, and how the pore permeability is regulated in different members of the AQP family.

Chromosomes must be accurately partitioned to daughter cells to prevent aneuploidy, a hallmark of many tumors and birth defects. Kinetochores are the macromolecular machines that segregate chromosomes by maintaining load-bearing attachments to the dynamic tips of microtubules. Here, we present the structure of isolated budding-yeast kinetochore particles, as visualized by EM and electron tomography of negatively stained preparations. The kinetochore appears as an  126-nm particle containing a large central hub surrounded by multiple outer globular domains. In the presence of microtubules, some particles also have a ring that encircles the microtubule. Our data, showing that kinetochores bind to microtubules via multivalent attachments, lay the foundation to uncover the key mechanical and regulatory mechanisms by which kinetochores control chromosome segregation and cell division.

Centrin is a calcium binding protein (CaBP) belonging to the EF-hand superfamily. As with other proteins within this family, centrin is a calcium sensor with multiple biological target proteins. We chose to study Chlamydomonas reinhardtii centrin (Crcen) and its interaction with melittin (MLT) as a model for CaBP complexes due to its amphipathic properties. Our goal was to determine the molecular interactions that lead to centrin-MLT complex formation, their relative stability, and the conformational changes associated with the interaction, when compared to the single components. For this, we determined the thermodynamic parameters that define Crcen-MLT complex formation. Two-dimensional infrared (2D IR) correlation spectroscopy were used to study the amide I', I'*, and side chain bands for (13)C-Crcen, MLT, and the (13)C-Crcen-MLT complex. This approach resulted in the determination of MLT's increased helicity, while centrin was stabilized within the complex. Herein we provide the first complete molecular description of centrin-MLT complex formation and the dissociation process. Also, discussed is the first structure of a CaBP-MLT complex by X-ray crystallography, which shows that MLT has a different binding orientation than previously characterized centrin-bound peptides. Finally, all of the experimental results presented herein are consistent with centrin maintaining an extended conformation while interacting with MLT. The molecular implications of these results are: (1) the recognition of hydrophobic contacts as requirements for initial binding, (2) minimum electrostatic interactions within the C-terminal end of the peptide, and (3) van der Waals interactions within MLTs N-terminal end are required for complex formation.

Pyrococcus furiosus phosphoglucose isomerase (PfPGI) is a metal-containing enzyme that catalyses the interconversion of glucose 6-phosphate (G6P) and fructose 6-phosphate (F6P). The recent structure of PfPGI has confirmed the hypothesis that the enzyme belongs to the cupin superfamily and identified the position of the active site. This fold is distinct from the alphabetaalpha sandwich fold commonly seen in phosphoglucose isomerases (PGIs) that are found in bacteria, eukaryotes and some archaea. Whilst the mechanism of the latter family is thought to proceed through a cis-enediol intermediate, analysis of the structure of PfPGI in the presence of inhibitors has led to the suggestion that the mechanism of this enzyme involves the metal-dependent direct transfer of a hydride between C1 and C2 atoms of the substrate. To gain further insight in the reaction mechanism of PfPGI, the structures of the free enzyme and the complexes with the inhibitor, 5-phospho-d-arabinonate (5PAA) in the presence and absence of metal have been determined. Comparison of these structures with those of equivalent complexes of the eukaryotic PGIs reveals similarities at the active site in the disposition of possible catalytic residues. These include the presence of a glutamic acid residue, Glu97 in PfPGI, which occupies the same position relative to the inhibitor as that of the glutamate that is thought to function as the catalytic base in the eukaryal-type PGIs. These similarities suggest that aspects of the catalytic mechanisms of these two structurally unrelated PGIs may be similar and based on an enediol intermediate.

Understanding cortical circuits will require mapping the connections between specific populations of neurons, as well as determining the dendritic locations where the synapses occur. The dendrites of individual cortical neurons overlap with numerous types of local and long-range excitatory axons, but axodendritic overlap is not always a good predictor of actual connection strength. Here we developed an efficient channelrhodopsin-2 (ChR2)-assisted method to map the spatial distribution of synaptic inputs, defined by presynaptic ChR2 expression, within the dendritic arborizations of recorded neurons. We expressed ChR2 in two thalamic nuclei, the whisker motor cortex and local excitatory neurons and mapped their synapses with pyramidal neurons in layers 3, 5A and 5B (L3, L5A and L5B) in the mouse barrel cortex. Within the dendritic arborizations of L3 cells, individual inputs impinged onto distinct single domains. These domains were arrayed in an orderly, monotonic pattern along the apical axis: axons from more central origins targeted progressively higher regions of the apical dendrites. In L5 arborizations, different inputs targeted separate basal and apical domains. Input to L3 and L5 dendrites in L1 was related to whisker movement and position, suggesting that these signals have a role in controlling the gain of their target neurons. Our experiments reveal high specificity in the subcellular organization of excitatory circuits.

In the hippocampus, the classical pyramidal cell type of the subiculum acts as a primary output, conveying hippocampal signals to a diverse suite of downstream regions. Accumulating evidence suggests that the subiculum pyramidal cell population may actually be comprised of discrete subclasses. Here, we investigated the extent and organizational principles governing pyramidal cell heterogeneity throughout the mouse subiculum. Using single-cell RNA-seq, we find that the subiculum pyramidal cell population can be deconstructed into eight separable subclasses. These subclasses were mapped onto abutting spatial domains, ultimately producing a complex laminar and columnar organization with heterogeneity across classical dorsal-ventral, proximal-distal, and superficial-deep axes. We further show that these transcriptomically defined subclasses correspond to differential protein products and can be associated with specific projection targets. This work deconstructs the complex landscape of subiculum pyramidal cells into spatially segregated subclasses that may be observed, controlled, and interpreted in future experiments.

Spinal muscular atrophy (SMA) results from reduced levels of the survival of motor neuron (SMN) protein, which has a well characterized function in spliceosomal small nuclear ribonucleoprotein assembly. Currently, it is not understood how deficiency of a housekeeping protein leads to the selective degeneration of spinal cord motor neurons. Numerous studies have shown that SMN is present in neuronal processes and has many interaction partners, including mRNA-binding proteins, suggesting a potential noncanonical role in axonal mRNA metabolism. In this study, we have established a novel technological approach using bimolecular fluorescence complementation (BiFC) and quantitative image analysis to characterize SMN-protein interactions in primary motor neurons. Consistent with biochemical studies on the SMN complex, BiFC analysis revealed that SMN dimerizes and interacts with Gemin2 in nuclear gems and axonal granules. In addition, using pull down assays, immunofluorescence, cell transfection, and BiFC, we characterized a novel interaction between SMN and the neuronal mRNA-binding protein HuD, which was dependent on the Tudor domain of SMN. A missense mutation in the SMN Tudor domain, which is known to cause SMA, impaired the interaction with HuD, but did not affect SMN axonal localization or self-association. Furthermore, time-lapse microscopy revealed SMN cotransport with HuD in live motor neurons. Importantly, SMN knockdown in primary motor neurons resulted in a specific reduction of both HuD protein and poly(A) mRNA levels in the axonal compartment. These findings reveal a noncanonical role for SMN whereby its interaction with mRNA-binding proteins may facilitate the localization of associated poly(A) mRNAs into axons.