Autophagy is an enigmatic cellular process in which double-membrane compartments, called "autophagosomes, form de novo adjacent to the endoplasmic reticulum (ER) and package cytoplasmic contents for delivery to lysosomes. Expansion of the precursor membrane phagophore requires autophagy-related 2 (ATG2), which localizes to the PI3P-enriched ER-phagophore junction. We combined single-particle electron microscopy, chemical cross-linking coupled with mass spectrometry, and biochemical analyses to characterize human ATG2A in complex with the PI3P effector WIPI4. ATG2A is a rod-shaped protein that can bridge neighboring vesicles through interactions at each of its tips. WIPI4 binds to one of the tips, enabling the ATG2A-WIPI4 complex to tether a PI3P-containing vesicle to another PI3P-free vesicle. These data suggest that the ATG2A-WIPI4 complex mediates ER-phagophore association and/or tethers vesicles to the ER-phagophore junction, establishing the required organization for phagophore expansion via the transfer of lipid membranes from the ER and/or the vesicles to the phagophore.

Significance Prokaryotes have adaptive immune systems that rely on CRISPRs (clustered regularly interspaced short palindromic repeats) and diverse CRISPR-associated ( cas ) genes. Cas1 and Cas2 are conserved components of CRISPR systems that are essential for integrating fragments of foreign DNA into CRISPR loci. In type I-F immune systems, the Cas2 adaptation protein is fused to the Cas3 interference protein. Here we show that the Cas2/3 fusion protein from Pseudomonas aeruginosa stably associates with the Cas1 adaptation protein, forming a 375-kDa propeller-shaped Cas1–2/3 complex. We show that Cas1, in addition to being an essential adaptation protein, also functions as a repressor of Cas2/3 nuclease activity and that foreign DNA binding by the CRISPR RNA-guided surveillance complex activates the Cas2/3 nuclease.

Summary The modulation of ion channel activity by lipids is increasingly recognized as a fundamental component of cellular signaling. The mucolipin transient receptor potential (TRPML) channel family belongs to the TRP superfamily1,2 and is composed of three members, TRPML1-3. TRPMLs are the major Ca2+-permeable channels on late endosomes and lysosomes (LEL). They regulate organelle Ca2+ releases important for various physiological processes, including organelle trafficking and fusion3. Loss-of-function mutations in the TRPML1 gene cause the neurodegenerative lysosomal storage disorder mucolipidosis IV (ML-IV), and a gain-of-function mutation in TRPML3 (Ala419Pro) gives rise to the Varitint-Waddler (Va) mouse phenotype4–6. Notably, TRPMLs are activated by the low-abundance and LEL-enriched signaling lipid PI(3,5)P2, while other phosphoinositides such as PI(4,5)P2, enriched in plasma membranes, inhibit TRPMLs7,8. Conserved basic residues at the N-terminus of the channels are important for PI(3,5)P2 activation and PI(4,5)P2 inhibition8. However, due to a lack of structural information, the mechanism by which TRPML channels recognize PI(3,5)P2 and increase its Ca2+ conductance remains elusive. Here we present the cryo-electron microscopy (cryo-EM) structure of a full-length TRPML3, at an average resolution of 2.9 Å. Our structure reveals not only the molecular basis of ion conduction but also the unique architecture of TRPMLs, wherein the voltage sensor-like domain is linked to the pore via a cytosolic domain we term the “mucolipin domain” (MLD). Combined with functional studies, we suggest that the MLD is responsible for PI(3,5)P2 binding and subsequent channel activation, and that it acts as a ‘gating pulley’ for lipid-dependent TRPML gating.

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Mammalian circadian rhythms are generated by a transcription-based feedback loop in which CLOCK:BMAL1 drives transcription of its repressors (PER1/2, CRY1/2), which ultimately interact with CLOCK:BMAL1 to close the feedback loop with ~24 hr periodicity. Here we pinpoint a key difference between CRY1 and CRY2 that underlies their differential strengths as transcriptional repressors. Both cryptochromes bind the BMAL1 transactivation domain similarly to sequester it from coactivators and repress CLOCK:BMAL1 activity. However, we find that CRY1 is recruited with much higher affinity to the PAS domain core of CLOCK:BMAL1, allowing it to serve as a stronger repressor that lengthens circadian period. We discovered a dynamic serine-rich loop adjacent to the secondary pocket in the photolyase homology region (PHR) domain that regulates differential binding of cryptochromes to the PAS domain core of CLOCK:BMAL1. Notably, binding of the co-repressor PER2 remodels the serine loop of CRY2, making it more CRY1-like and enhancing its affinity for CLOCK:BMAL1.

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A key player in the intracellular trafficking network is cytoplasmic dynein, a protein complex that transports molecular cargo along microtubule tracks. It has been shown that vertebrate dynein's movement becomes strikingly enhanced upon interacting with a cofactor named dynactin and one of several cargo-adapters, such as BicaudalD2. However, the mechanisms responsible for this increase in transport efficiency are not well understood, largely due to a lack of structural information. We used cryo-electron tomography to visualize the first 3-dimensional structure of the intact dynein-dynactin complex bound to microtubules. Our structure reveals that the dynactin-cargo-adapter complex recruits and binds to two dimeric cytoplasmic dyneins. Interestingly, the dynein motor organization closely resembles that of axonemal dynein, suggesting that cytoplasmic dynein and axonemal dyneins may utilize similar mechanisms to coordinate multiple motors. We propose that grouping dyneins onto a single dynactin scaffold promotes collective force production as well as unidirectional processive motility. These findings provide a structural platform that facilitates a deeper biochemical and biophysical understanding of dynein regulation and cellular transport.