Vesicle docking and fusion pore modulation by the neuronal calcium sensor Synaptotagmin-1.

Synaptotagmin-1 (Syt1) is a major calcium sensor for rapid neurotransmitter release in neurons and hormone release in many neuroendocrine cells. It possesses two tandem cytosolic C2 domains that bind calcium, negatively charged phospholipids, and the neuronal SNARE complex. Calcium binding to Syt1 triggers exocytosis, but how this occurs is not well understood.

Vesicle docking and fusion pore modulation by the neuronal calcium sensor Synaptotagmin-1.

Synaptotagmin-1 (Syt1) is a major calcium sensor for rapid neurotransmitter release in neurons and hormone release in many neuroendocrine cells. It possesses two tandem cytosolic C2 domains that bind calcium, negatively charged phospholipids, and the neuronal SNARE complex. Calcium binding to Syt1 triggers exocytosis, but how this occurs is not well understood.

Cellular function of the GndA small open reading frame-encoded polypeptide during heat shock.

Over the past 15 years, hundreds of previously undiscovered bacterial small open reading frame (sORF)-encoded polypeptides (SEPs) of fewer than fifty amino acids have been identified, and biological functions have been ascribed to an increasing number of SEPs from intergenic regions and small RNAs. However, despite numbering in the dozens in , and hundreds to thousands in humans, same-strand nested sORFs that overlap protein coding genes in alternative reading frames remain understudied. In order to provide insight into this enigmatic class of unannotated genes, we characterized GndA, a 36-amino acid, heat shock-regulated SEP encoded within the +2 reading frame of the gene in K-12 MG1655.

Detection of membrane fission in single Bacillus subtilis cells during endospore formation with high temporal resolution.

Membrane fission is an essential process in all domains of life. The underlying mechanisms remain poorly understood in bacteria, partly because suitable assays are lacking. Here, we describe an assay to detect membrane fission during endospore formation in single Bacillus subtilis cells with a temporal resolution of ∼1 min.

Membrane fission during bacterial spore development requires cellular inflation driven by DNA translocation.

Bacteria require membrane fission for both cell division and endospore formation. In Bacillus subtilis, sporulation initiates with an asymmetric division that generates a large mother cell and a smaller forespore that contains only a quarter of its genome. As the mother cell membranes engulf the forespore, a DNA translocase pumps the rest of the chromosome into the small forespore compartment, inflating it due to increased turgor.

Polybasic Patches in Both C2 Domains of Synaptotagmin-1 Are Required for Evoked Neurotransmitter Release.

Synaptotagmin-1 (Syt1) is a vesicular calcium sensor required for synchronous neurotransmitter release, composed of a single-pass transmembrane domain linked to two C2 domains (C2A and C2B) that bind calcium, acidic lipids, and SNARE proteins that drive fusion of the synaptic vesicle with the plasma membrane. Despite its essential role, how Syt1 couples calcium entry to synchronous release is poorly understood. Calcium binding to C2B is critical for synchronous release, and C2B additionally binds the SNARE complex.

Rapid propagation of membrane tension at retinal bipolar neuron presynaptic terminals.

Many cellular activities, such as cell migration, cell division, phagocytosis, and exo-endocytosis, generate and are regulated by membrane tension gradients. Membrane tension gradients drive membrane flows, but there is controversy over how rapidly plasma membrane flow can relax tension gradients. Here, we show that membrane tension can propagate rapidly or slowly, spanning orders of magnitude in speed, depending on the cell type.

FisB relies on homo-oligomerization and lipid binding to catalyze membrane fission in bacteria.

Little is known about mechanisms of membrane fission in bacteria despite their requirement for cytokinesis. The only known dedicated membrane fission machinery in bacteria, fission protein B (FisB), is expressed during sporulation in Bacillus subtilis and is required to release the developing spore into the mother cell cytoplasm. Here, we characterized the requirements for FisB-mediated membrane fission.

DNA-Origami-Based Fluorescence Brightness Standards for Convenient and Fast Protein Counting in Live Cells.

Fluorescence microscopy has been one of the most discovery-rich methods in biology. In the digital age, the discipline is becoming increasingly quantitative. Virtually all biological laboratories have access to fluorescence microscopes, but abilities to quantify biomolecule copy numbers are limited by the complexity and sophistication associated with current quantification methods.

Human ATG3 binding to lipid bilayers: role of lipid geometry, and electric charge.

Specific protein-lipid interactions lead to a gradual recruitment of AuTophaGy-related (ATG) proteins to the nascent membrane during autophagosome (AP) formation. ATG3, a key protein in the movement of LC3 towards the isolation membrane, has been proposed to facilitate LC3/GABARAP lipidation in highly curved membranes. In this work we have performed a biophysical study of human ATG3 interaction with membranes containing phosphatidylethanolamine, phosphatidylcholine and anionic phospholipids.

Human Atg8-cardiolipin interactions in mitophagy: Specific properties of LC3B, GABARAPL2 and GABARAP.

The phospholipid cardiolipin (CL) has been proposed to play a role in selective mitochondrial autophagy, or mitophagy. CL externalization to the outer mitochondrial membrane would act as a signal for the human Atg8 ortholog subfamily, MAP1LC3 (LC3). The latter would mediate both mitochondrial recognition and autophagosome formation, ultimately leading to removal of damaged mitochondria.

Lipid Geometry and Bilayer Curvature Modulate LC3/GABARAP-Mediated Model Autophagosomal Elongation.

Autophagy, an important catabolic pathway involved in a broad spectrum of human diseases, implies the formation of double-membrane-bound structures called autophagosomes (AP), which engulf material to be degraded in lytic compartments. How APs form, especially how the membrane expands and eventually closes upon itself, is an area of intense research. Ubiquitin-like ATG8 has been related to both membrane expansion and membrane fusion, but the underlying molecular mechanisms are poorly understood.

Minimalist Model Systems Reveal Similarities and Differences between Membrane Interaction Modes of MCL1 and BAK.

Proteins belonging to the BCL2 family are key modulators of apoptosis that establish a complex network of interactions among themselves and with other cellular factors to regulate cell fate. It is well established that mitochondrial membranes are the main locus of action of all BCL2 family proteins, but it is difficult to obtain a precise view of how BCL2 family members operate at the native mitochondrial membrane environment during apoptosis. Here, we used minimalist model systems and multiple fluorescence-based techniques to examine selected membrane activities of MCL1 and BAK under apoptotic-like conditions.

Lipid-dependent bimodal MCL1 membrane activity.

Increasing evidence indicates that the mitochondrial lipid membrane environment directly modulates the BCL2 family protein function, but the underlying mechanisms are still poorly understood. Here, we used minimalistic reconstituted systems to examine the influence of mitochondrial lipids on MCL1 activity and conformation. Site-directed mutagenesis and fluorescence spectroscopic analyses revealed that the BCL2 homology region of MCL1 (MCL1ΔNΔC) inhibits permeabilization of MOM-like membranes exclusively via canonical BH3-into-groove interactions with both cBID-like activators and BAX-like effectors.

Proapoptotic Bax and Bak proteins form stable protein-permeable pores of tunable size.

The Bcl-2 proapoptotic proteins Bax and Bak mediate the permeabilization of the mitochondrial outer membrane during apoptosis. Current models consider that Bax and Bak form pores at the mitochondrial outer membrane that are responsible for the release of cytochrome c and other larger mitochondrial apoptotic factors (i.e.

Reconstitution of proapoptotic BAK function in liposomes reveals a dual role for mitochondrial lipids in the BAK-driven membrane permeabilization process.

BAK is a key effector of mitochondrial outer membrane permeabilization (MOMP) whose molecular mechanism of action remains to be fully dissected in intact cells, mainly due to the inherent complexity of the intracellular apoptotic machinery. Here we show that the core features of the BAK-driven MOMP pathway can be reproduced in a highly simplified in vitro system consisting of recombinant human BAK lacking the carboxyl-terminal 21 residues (BAKΔC) and tBID in combination with liposomes bearing an appropriate lipid environment. Using this minimalist reconstituted system we established that tBID suffices to trigger BAKΔC membrane insertion, oligomerization, and pore formation.

Endophilin B1/Bif-1 stimulates BAX activation independently from its capacity to produce large scale membrane morphological rearrangements.

Endophilin B1/BAX-interacting factor 1 (Bif-1) is a protein that cooperates with dynamin-like protein 1 (DLP1/Drp1) to maintain normal mitochondrial outer membrane (MOM) dynamics in healthy cells and also contributes to BAX-driven MOM permeabilization (MOMP), the irreversible commitment point to cell death for the majority of apoptotic stimuli. However, despite its importance, exactly how Bif-1 fulfils its proapoptotic role is unknown. Here, we demonstrate that the stimulatory effect of Bif-1 on BAX-driven MOMP and on BAX conformational activation observed in intact cells during apoptosis can be recapitulated in a simplified system consisting of purified proteins and MOM-like liposomes.

BIM and tBID are not mechanistically equivalent when assisting BAX to permeabilize bilayer membranes.

BIM and tBID are two BCL-2 homology 3 (BH3)-only proteins with a particularly strong capacity to trigger BAX-driven mitochondrial outer membrane permeabilization, a crucial event in mammalian apoptosis. However, the means whereby BIM and tBID fulfill this task is controversial. Here, we used a reconstituted liposomal system bearing physiological relevance to explore systematically how the BAX-permeabilizing function is influenced by interactions of BIM/BID-derived proteins and BH3 motifs with multidomain BCL-2 family members and with membrane lipids.