Biophysical Society Conference | Estes Park 2023

Conferences

Membrane Fusion and Budding

Estes Park, Colorado | September 24–27, 2023

Organizing Committee

Arun Anantharam, University of Toledo, USA Michelle Knowles, University of Denver, USA Ling-Gang Wu, National Institutes of Health (NIH), USA

Thank You to Our Sponsors

Thank you to all sponsors for their support.

Membrane Fusion and Budding

Welcome Letter

September 2023

Dear Colleagues,

We would like to welcome you to the Biophysical Society Conference on Membrane Fusion and Budding . Our meeting will bring together scientists at the forefront of membrane fusion and budding, who use a wide range of methodologies and biological systems. Overall, this conference will feature approximately 60 posters, 42 lectures and bring together over 100 well recognized scientists from a wide range of backgrounds and expertise. We hope that this meeting will not only provide a place to share your recent findings, but also to help promote new collaborations, helpful discussions and future connections. We hope that you will all actively take part in the discussions following each talk, in the poster sessions, and in the informal exchanges that will be possible during the coffee breaks and meals. We also hope that you will also enjoy the beautiful mountain wilderness in Estes Park, Colorado! We would also like to thank our generous sponsors: NIH-National Institute of Neurological Disorders and Stroke, Abberior Instruments, Evident Scientific, and Nikon Instruments for supporting our meeting.

The Organizing Committee: Arun Anantharam Michelle Knowles Ling-Gang Wu

Membrane Fusion and Budding

Meeting Code of Conduct

Biophysical Society Code of Conduct, Anti-Harassment Policy The Biophysical Society (BPS) is committed to providing an environment that encourages the free expression and exchange of scientific ideas. As a global, professional Society, the BPS is committed to the philosophy of equal opportunity and respectful treatment for all, regardless of national or ethnic origin, religion or religious belief, gender, gender identity or expression, race, color, age, marital status, sexual orientation, disabilities, veteran status, or any other reason not related to scientific merit. All BPS meetings and BPS-sponsored activities promote an environment that is free of inappropriate behavior and harassment by or toward all attendees and participants of Society events, including speakers, organizers, students, guests, media, exhibitors, staff, vendors, and other suppliers. BPS expects anyone associated with an official BPS-sponsored event to respect the rules and policies of the Society, the venue, the hotels, and the city. Definition of Harassment The term “harassment” includes but is not limited to epithets, unwelcome slurs, jokes, or verbal, graphic or physical conduct relating to an individual’s race, color, religious creed, sex, national origin, ancestry, citizenship status, age, gender or sexual orientation that denigrate or show hostility or aversion toward an individual or group. Sexual harassment refers to unwelcome sexual advances, requests for sexual favors, and other verbal or physical conduct of a sexual nature. Behavior and language that are welcome/acceptable to one person may be unwelcome/offensive to another. Consequently, individuals must use discretion to ensure that their words and actions communicate respect for others. This is especially important for those in positions of authority since individuals with lower rank or status may be reluctant to express their objections or discomfort regarding unwelcome behavior. It does not refer to occasional compliments of a socially acceptable nature. It refers to behavior that is not welcome, is personally offensive, debilitates morale, and therefore, interferes with work effectiveness. The following are examples of behavior that, when unwelcome, may constitute sexual harassment: sexual flirtations, advances, or propositions; verbal comments or physical actions of a sexual nature; sexually degrading words used to describe an individual; a display of sexually suggestive objects or pictures; sexually explicit jokes; unnecessary touching. Attendees or participants who are asked to stop engaging in harassing behavior are expected to comply immediately. Anyone who feels harassed is encouraged to immediately inform the alleged harasser that the behavior is unwelcome. In many instances, the person is unaware that their conduct is offensive and when so advised can easily and willingly correct the conduct so that it does not reoccur. Anyone who feels harassed is NOT REQUIRED to address the person believed guilty of inappropriate treatment. If the informal discussion with the alleged harasser is unsuccessful in remedying the problem or if the complainant does not feel comfortable with such an approach, they can report the behavior as detailed below. Reported or suspected occurrences of harassment will be promptly and thoroughly investigated. Following an investigation, BPS will immediately take any necessary and appropriate action. BPS will not permit or condone any acts of retaliation against anyone who files harassment complaints or cooperates in the investigation of same. Reporting a Violation Violations of this Conduct Policy should be reported immediately. If you feel physically unsafe or believe a crime has been committed, you should report it to the police immediately. To report a violation to BPS:

• You may do so in person at the Annual Meeting at the BPS Business Office in the convention center.

Membrane Fusion and Budding

Meeting Code of Conduct

• You may do so in person to BPS senior staff at Thematic Meetings, BPS Conferences, or other BPS events.

• At any time (during or after an event), you can make a report through

http://biophysics.ethicspoint.com or via a dedicated hotline (phone numbers listed on the website) which will collect and relay information in a secure and sensitive manner.

Reported or suspected occurrences of harassment will be promptly and thoroughly investigated per the procedure detailed below. Following an investigation, BPS will immediately take any necessary and appropriate action. BPS will not permit or condone any acts of retaliation against anyone who files harassment complaints or cooperates in the investigation of same. Investigative Procedure All reports of harassment or sexual harassment will be treated seriously. However, absolute confidentiality cannot be promised nor can it be assured. BPS will conduct an investigation of any complaint of harassment or sexual harassment, which may require limited disclosure of pertinent information to certain parties, including the alleged harasser. Once a complaint of harassment or sexual harassment is received, BPS will begin a prompt and thorough investigation. Please note, if a complaint is filed anonymously, BPS may be severely limited in our ability to follow-up on the allegation. • An impartial investigative committee, consisting of the current President, President-Elect, and Executive Officer will be established. If any of these individuals were to be named in an allegation, they would be excluded from the committee. • The committee will interview the complainant and review the written complaint. If no written complaint exists, one will be requested. • The committee will speak to the alleged offender and present the complaint. • The alleged offender will be given the opportunity to address the complaint, with sufficient time to respond to the evidence and bring his/her own evidence. • If the facts are in dispute, the investigative team may need to interview anyone named as witnesses. • The investigative committee may seek BPS Counsel’s advice. • Once the investigation is complete, the committee will report their findings and make recommendations to the Society Officers. • If the severity of the allegation is high, is a possible repeat offense, or is determined to be beyond BPS’s capacity to assess claims and views on either side, BPS may refer the case to the alleged offender’s home institution (Office of Research Integrity of similar), employer, licensing board, or law enforcement for their investigation and decision. Disciplinary Actions Individuals engaging in behavior prohibited by this policy as well as those making allegations of harassment in bad faith will be subject to disciplinary action. Such actions range from a written warning to ejection from the meeting or activity in question without refund of registration fees, being banned from participating in future Society meetings or Society-sponsored activities, being expelled from membership in the Society, and reporting the behavior to their employer or calling the authorities. In the event that the individual is dissatisfied with the results of the investigation, they may appeal to the President of the Society. Any questions regarding this policy should be directed to the BPS Executive Officer or other Society Officer.

Membrane Fusion and Budding

Table of Contents

Table of Contents

General Information……………………………………………………………………………....1 Program Schedule..……………………………………………………………………………….3 Speaker Abstracts………………………………………………………………………………...9 Poster Sessions…………………………………………………………………………………...51

Membrane Fusion and Budding

General Information

GENERAL INFORMATION

Registration/Information Location and Hours Venue check-in to obtain your room key will be located at the Front Desk at Ridgeline Hotel Estes Park, 101 South Saint Vrain Avenue, Estes Park, CO 80517. The BPS Registration Desk, to pick up your badge and meeting materials, will be located in the Ballroom Front Foyer during the following times: Sunday, September 24 5:00 PM – 8:45 PM Monday, September 25 8:00 AM – 5:00 PM Tuesday, September 26 8:30 AM – 4:15 PM Wednesday, September 27 8:30 AM – 4:30 PM Instructions for Presentations (1) Presentation Facilities: A data projector will be available in the Ballroom Salon ABC. Speakers are required to bring their own laptops and adaptors. It is recommended to have a backup of the presentation on a USB drive in case of any unforeseen circumstances. Speakers are advised to preview their final presentations before the start of each session. (2) Poster Session: 1) All poster sessions will be held in the Ballroom Front Foyer. 2) A display board measuring 5 feet wide (152.4 cm) and 3.4 feet tall (103.6 cm) will be provided for each poster. Poster boards are numbered according to the same numbering scheme as listed in the E-book. 3) Poster boards require pushpins or thumbtacks for mounting. Authors are expected to bring their own mounting materials. 4) There will be formal poster presentations on Monday, Tuesday, and Wednesday. Poster presentation times vary by day, so please refer to the daily schedule for your formal presentation date and time. Ninety (90) minutes have been allotted for poster presentations each day. Presenting authors with odd-numbered poster boards should present during the first 45 minutes, and those with even-numbered poster boards should present during the last 45 minutes. 5) During the assigned poster presentation sessions, presenters are requested to remain in front of their poster boards to meet with attendees. 6) All posters left uncollected at the end of the meeting will be discarded.

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Membrane Fusion and Budding

General Information

Note Pads/Pens Society pens will be provided, however please bring your own note pad. Covid Precautions

While there are no formal covid protocols, it is suggested that you should test in advance, stay home if you are ill, and isolate and test if you experience symptoms at the meeting. Masks are not mandatory but encouraged for all indoor spaces. Meals, Coffee Breaks, and Socials Breakfasts and luncheons will be served in Ballroom Salon D-F. Wednesday evening’s dinner will also be held in Ballroom Salon D-F. Coffee Breaks will be held in the Front Foyer. The evening socials scheduled for Sunday-Tuesday will be held in the Latitude 105 Bar. Smoking Please be advised that the Ridgeline Hotel Estes Park is a non-smoking facility. Name Badges Name badges will be given to you when you check-in at the Registration Desk in the Ballroom Front Foyer. Badges are required to enter all scientific sessions, poster sessions, and social functions. Please wear your badge throughout the conference. Internet Wi-Fi will be provided at the venue. On-Site Contact Information If you have any further requirements during the meeting, please contact the meeting staff at the registration desk from September 24-27 during registration hours. In case of emergency, you may contact the following: Dorothy Chaconas Phone : 301-785-0802 Email: dchaconas@biophysics.org Erica Bellavia Phone : 571-435-7669 Email: ebellavia@biophysics.org

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Membrane Fusion and Budding

Daily Schedule

Membrane Fusion and Budding Estes Park, Colorado September 24-27, 2023 All scientific sessions will be held in the Ballroom ABC unless otherwise noted. PROGRAM

Sunday, September 24, 2023

5:00 PM – 8:45 PM

Registration/Information

Ballroom Front Foyer

7:00 PM – 7:10 PM

Welcome and Opening Remarks Ling- Gang Wu, NIH, USA

Session I

Keynotes I

7:10 PM – 7:35 PM

John Heuser, Washington University in St. Louis, USA Revisiting Electron Microscopy’s “Golden Decade” of Imaging Synaptic Vesicle Dynamics: the 1970’s Edwin Chapman, University of Wisconsin-Madison, USA Unexpected Interplay Between Two Presynaptic Calcium Sensors James Rothman, Yale University, USA Turbo-Charging Synaptic Vesicles for Explosive Release of Neurotransmitters

7:35 PM – 8:00 PM

8:00 PM – 8:45 PM

8:45 PM – 10:30 PM

Social Gathering

Latitude 105 Bar

Monday, September 25, 2023 7:30 AM – 8:30 AM Breakfast

Ballroom DEF

8:00 AM – 5:00 PM

Registration/Information

Ballroom Front Foyer

Session II

Dense Core Vesicle Fusion and Fission Chair: Volker Kiessling, University of Virginia, USA

8:30 AM – 8:55 AM

Erdem Karatekin, Yale University, USA Membrane Fusion and Fission: Lessons from Unexpected Places Kobina Essandoh, University of Michigan, USA* Molecular Regulation of Atrial Natriueretic Peptide Secretion in Cardiomyocytes by the RAB3GAP1 and the RAB3A GTPASE Cycle Arun Anantharam, The University of Toledo, USA Signaling Networks Coupling Calcium to Secretion in the Adrenal Medulla

8:55 AM – 9:10 AM

9:10 AM – 9:35 AM

9:35 AM – 10:00 AM

Ira Milosevic, Oxford University, United Kingdom Negative Regulation of Exocytosis by Amisyn

10:00 AM – 10:30 AM

Break

Ballroom Front Foyer

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Daily Schedule

Session III

Intra/Inter-Cellular Fusion and Fission Chair: TBD

10:30 AM – 10:55 AM

Aurelian Roux, University of Geneva, Switzerland Reconstituting ESCRT-III Mediated Membrane Fission In Vitro Sandy Maday, University of Pennsylvania, USA Autophagy-Lysosomal Pathways in Neurons and Astrocytes Michael Kozlov, Tel Avi University, Israel Model for Ring Collapse and Membrane Fission in ER Tubular Networks Lisa Redlingshofer, Max Planck Institute, Germany* Reconstructing the Collective Behaviour of the Endosomal Membrane Fusion Machinery Elizabeth Chen, University of Texas, Southwestern Medical Center, USA Function of ABC G1/G4 Transporters and Cholesterol in Cell-Cell Fusion Thomas Pucadyil, Indian Institute of Science and Education and Research, India Mechanistic Analysis of Membrane Fission and Discovery of Novel Fission Proteins David Drubin, University of California, Berkeley, USA Harnessing Actin Assembly Forces to Drive Vesicle Formations During Clathrin-Mediated Endocytosis Richard Hooy, University of California, Berkeley, USA* Self-Assembly and Structure of a Clathrin-Independent AP-1: ARF1 Tubular Membrane Coat Vadim Frolov, Ikerbasque- Basque Foundation for Science, Spain Molecular Mechanosensing in Dynamin-Driven Membrane Remodeling Extracellular and Mitochondrial Vesicles Chair: Prabhodh Abbineni, Loyola University, Chicago, USA Chantell Evans, Duke University, USA Mitochondrial Derived Vesicles: A New Mitochondrial Quality Control Mechanism Alissa Weaver, Vanderbilt University, USA Biogenesis of RNA-Containing Extracellular Vesicles at ER-Membrane Contact Sites Lunch Endocytosis, Membrane Bending and Fission Chair: Sarah Nyenhuis, NIH, USA Poster Session I Free Time/Dinner on Own

10:55 AM – 11:20 AM

11:20 AM – 11:45 AM

11:45 AM – 12:00 PM

12:00 PM – 12:25 PM

12:25 PM – 1:25 PM

Ballroom DEF

Session IV

1:35 PM – 2:00 PM

2:00 PM – 2:25 PM

2:25 PM – 2:40 PM

2:40 PM – 3:05PM

3:15 PM – 4:45 PM

Ballroom Front Foyer

4:45 PM – 7:30 PM

Session V

7:30 PM – 7:55 PM

7:55 PM – 8:20 PM

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Membrane Fusion and Budding

Daily Schedule

8:20 PM – 8:45 PM

Ann Wehman, University of Denver, USA Extracellular Vesicles Budding and Lipid Asymmetry

8:45 PM – 9:10 PM

Michelle Knowles, University of Denver, USA Multivesicular Endosome Fusion Kinetics Depend on Exosome Tethering and Temperature

9:10 PM – 10:30 PM

Social Gathering

Latitude 105 Bar

Tuesday, September 26, 2023 7:30 AM – 8:30 AM Breakfast

Ballroom DEF

8:30 AM – 4:15 PM

Registration/Information

Ballroom Front Foyer

Session VI

Synaptic Vesicle Fusion Chair: Jianyuan Sun, Chinese Academy of Sciences, China

8:30 AM – 8:55 AM

Ege Kavalali, Vanderbilt University, USA Functional Nano-Organization of Synapses

8:55 AM – 9:10 AM

Amelia Ralowicz, Dartmouth College, USA* Spontaneous Synaptic Vesicle Function is Correlated with Number of Vesicles in Readily Releasable Pool Jeremy Dittman , Cornell University, Weill Medical College, USA Molecular Insights into Munc13 and Complexin Function at the Synapse Skyler Jackman, Oregon Health and Science University, USA High-Affinity Calcium Sensors Mediate Asynchronous Vesicle Fusion

9:10 AM – 9:35 AM

9:35 AM – 10:00 AM

10:00 AM – 10:30 AM

Coffee Break

Ballroom Front Foyer

Session VI I

Fusion and Fission in Infection and Disease Chair: TBD

10:30 AM – 10:55 AM

Carole Parent, University of Michigan, USA The Neutrophil Nucleus as a Signaling Hub During Neutrophil Chemotaxis Gregory Melikian, Emory University, USA HIV-1 Preferentially Fuses with PH-Neutral Vesicles in Cell Lines and Primary CD4 + T-Cells

10:55 AM – 11:20 AM

11:20 AM – 11:35 AM

Xin Wang, NIH* Macropinocytosis in Sars CoV2 Infection

11:35 AM – 11:50 AM

Gonzalo Gonzales-Del Pino, Tufts University, USA* Structural and Functional Characterization of the Herpes Simplex Virus Membrane Fusion Regulator, GH/GL Comert Kural , Ohio State University, USA Sensitization of Cancer Cells to FAS-Induced Apoptosis Through Endocytosis Inhibition

11:50 AM – 12:15 PM

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Membrane Fusion and Budding

Daily Schedule

12 :15 PM – 1:15 PM

Lunch

Ballroom DEF

Session VIII

Membrane Bending, Fusion, and Endocytosis Chair: Ioana Butu, Columbia University, USA

1:25 PM – 1:50 PM

Wenting Zhao, Nanyang Technological University, Singapore Nanoscale Membrane Topography Engineering: In Cells, In Vitro and In Between Ben O’Shaugnessy, Columbia University, USA Mechanical Regulation of Membrane Fusion, Exocytosis and Endocytosis Studied by Coarse-Grained Simulation and Experimental Data Analysis Aline Tschanz, EMBL, Germany* Clathrin Coats Partially Preassemble and Subsequently Bend During Endocytosis Marko Kaksonen, University of Geneva, Switzerland Evolutionary Cell Biology Approach to Clathrin-Mediated Endocytosis Dense Core Vesicle Fusion and Endocytosis Chair: Debasis Das, Tata Institute of Fundamental Research, India Ling-Gang Wu, NIH, NINDS, USA Dynamics of Membrane Fusion and Endocytosis in Secretory Cells Lukas Tamm, University of Virginia, USA Membrane Lipids Couple Synaptotagmin to SNARE Mediated Granule Fusion in Insulin Secreting Cells Manfred Lindau, University of Miami, USA All SNAP25 Copies in the Vesicle-Plasma Membrane Contact Zone Change Conformation During Vesicle Priming Corey Smith, Case Western Reserve University, USA Rapid Electrochemical Measure of Neurotransmitter Dynamics in a Beating Pig Heart Poster Session II Free Time/Dinner on Own

1:50 PM – 2:15 PM

2:15 PM – 2:30 PM

2:30 PM – 2:55 PM

3:05 PM – 4:35 PM

Ballroom Front Foyer

4:35 PM – 7:30 PM

Session IV

7:30 PM – 7:55 PM

7:55 PM – 8:20 PM

8:20 PM – 8:45 PM

8 :45 PM – 9 :10 PM

Social Gathering

Latitude 105 Bar

9:10 PM – 10:30 PM

Wednesday, September 27, 2023

7:30 AM – 8:30 AM

Breakfast

Ballroom DEF

8:30 AM – 4:30 PM

Registration/Information

Ballroom Front Foyer

Session X

Endocytosis and Membrane Bending

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Membrane Fusion and Budding

Daily Schedule

Chair: Jefferson Knight, University of Colorado, Denver, USA

8:30 AM – 8:45 AM

Santiago Di Pietro, Colorado State University, USA* Mechanism of Actin Capping Protein Recruitment and Turnover During Clathrin Mediated Endocytosis Justin Taraska, NIH, USA Measuring the Nanoscale Structure of Endocytosis with Light and Electron Microscopy Margaret Johnson, Johns Hopkins University, USA Membrane Bending and Assembly Costs for Clathrin Coat Formation

8:45 AM – 9:10 AM

9:10 AM – 9:35 AM

9:35 AM – 10:00 AM

Shigeki Watanabe, Johns Hopkins University, USA Dynamin Prime – Accelerating Endocytosis at Synapses

10:00 AM – 10:30 AM

Coffee Break

Ballroom Front Foyer

Session XI

Synaptic Vesicle Fusion and Endocytosis Chair: Jiajie Diao, University of Cincinnati, USA

10:30 AM – 10:55 AM

Noa Lipstien-Thoms, Max Planck Institute for Multidisciplinary Sciences, Germany Human UNC13A Gene Variations Cause a Neurodevelopmental Syndrome and Impair Synaptic Function Stephanie Gupton, University of North Carolina at Chapel Hill, USA Spatial and Temporal Regulation of Constitutive Exocytosis in Cell Shape Change Michael Cousin, University of Edinburgh, United Kingdom Dynamin-1 Dysfunction in Human Disease Stephane Gasman, Institut des Neurosciences Cellulaire et Intégratives, CNRS, France* Transbilayer Phospholipid Scrambling is Essential for Efficient Synaptic Vesicle Endocytosis

10:55 AM – 11:20 AM

11:20 AM – 11:45 AM

11:45 PM – 12:00 PM

12:00 PM – 1:00 PM

Lunch

Ballroom DEF

Session XII

Fusion Pore and Release Mechanisms Chair: Ronald Holz, University of Michigan, USA

1:10 PM – 1:35 PM

Zhuan Zhou, Peking University, China Differential Co-Release of Catecholamine and ATP from a Vesicle Fusion Pore in Mouse Adrenal Chromaffin Cells Joshua Zimmerberg, NIH, USA Aggregation-Dependent Lipid/Fusion Peptide Pore Formation, and Microvesicles that Secrete Exosomes Meyer Jackson, University of Wisconsin-Madison, USA Fusion Pore Dynamics in Endocrine and Synaptic Exocytosis

1:35 PM – 2:00 PM

2:00 PM – 2:25 PM

2:25 PM – 2:50 PM

Sebastian Barg, Uppsala University, Sweden Fusion Pore Regulation in Insulin-Secreting Beta Cells

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Membrane Fusion and Budding

Daily Schedule

3:00 PM – 4:30 PM

Poster Session III

Ballroom Front Foyer

4:30 PM – 5:15 PM

Free Time

Session XIII

Keynotes II Chair: Michelle Knowles, University of Denver, USA Phyllis Hanson, University of Michigan, USA Beyond Repair: ESCRT’s Enhance Membrane Resilience

5:15 PM – 5:40 PM

5:40 PM – 6:05 PM

Jenny Hinshaw, NIH, USA Cryo-EM Structures of Dynamin Proteins Involved in Membrane Fission and Fusion

6:05 PM – 6:50 PM

Axel Brunger, Stanford University, USA Molecular Mechanisms of Calcium-Triggered Exocytosis

6:50 PM – 7:00 PM

Closing Remarks and Biophysical Journal Poster Awards

7:30 PM

Dinner

Ballroom DEF

*Contributed talks selected from among submitted abstracts

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Membrane Fusion and Budding

Speaker Abstracts

SPEAKER ABSTRACTS

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Sunday Speaker Abstracts

REVISITING ELECTRON MICROSCOPY’S “GOLDEN DECADE” OF IMAGING SYNAPTIC VESICLE DYNAMICS: THE 1970’S John Heuser 1 ; 1 Washington University in St. Louis, St. Louis, MO, USA No Abstract

SYNAPTOTAGMIN 7 DOCKS SYNAPTIC VESICLES FOR DOC2Α -TRIGGERED ASYNCHRONOUS NEUROTRANSMITTER RELEASE Edwin R. Chapman 1 ; Zhenyong Wu 2 ; Grant F. Kusick 3 ; Shigeki Watanabe 3 ; 1 HHMI & University of Wisconsin-Madison, Neuroscience, Madison, WI, USA 2 Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai, China 3 Johns Hopkins University, School of Medicine, Baltimore, MD, USA The molecular basis of asynchronous neurotransmitter release remains enigmatic despite decades of intense study. Synaptotagmin (syt) 7 and Doc2 have both been proposed as Ca 2+ sensors that trigger this mode of exocytosis, but conflicting findings have led to controversy. Here, we demonstrate that at excitatory mouse hippocampal synapses from cultured neurons and acute slices, Doc2α is the major Ca 2+ sensor for asynchronous release, while syt7 supports this process through activity-dependent docking of synap tic vesicles. In synapses lacking Doc2α, asynchronous release after single action potentials is strongly reduced, while deleting syt7 has no effect. However, in the absence of syt7, docked vesicles cannot recover on millisecond timescales. Consequently, both synchronous and asynchronous release depress from the second pulse on during repetitive activity. By contrast, synapses lacking Doc2α have normal activity dependent docking, but continue to exhibit decreased asynchronous release after multiple stimuli. Moreover, disruption of both Ca 2+ sensors is non-additive. These findings result in a new model whereby syt7 drives activity-dependent docking, thus ‘feeding’ synaptic vesicles to Doc2 for asynchronous release during ongoing transmission.

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Membrane Fusion and Budding

Sunday Speaker Abstracts

TURBO-CHARGING SYNAPTIC VESICLES FOR EXPLOSIVE RELEASE OF NEUROTRANSMITTERS

James E Rothman 1 ; 1 Yale University, Department of Cell Biology, New Haven, CT, USA

How neurotransmitter release occurs thousands of times faster than other forms of SNARE dependent membrane fusion has been a vexing problem. We have now discovered a simple principle that can explain this involving two layers of SNAREpins. A ring of 6 "central" SNAREpins is released by Ca++ to open the fusion pore. Unlike fusion reactions, we suggest that synaptic vesicles are "turbo-charged" by an outer set of 6 "peripheral SNAREpins" that provides additional force. This model has its origin in cryo-EM tomography that revealed a protein structure with 6-fold symmetry at the interface of each ready-release synaptic vesicle with the active zone plasma membrane. Our current studies involve single molecule counting of SNAREpins in single synthetic ready-release vesicles (using fully-defined reconstitutions of vesicles with suspended bilayers) and novel high resolution cryo-EM structures of the membrane-bound SNARE-assembling chaperone Munc13. Together, the new results suggest that the observed 6-fold symmetry results from the de novo assembly of a series of sequential, symmetrical supra-molecular machines. In this model, the vesicle is initially captured by a hexagonal "basket" of 18 copies of the chaperone Munc13 in their upright conformation. Munc13 then transits thru a lateral hexagon arrangement, moving the vesicle closer to the plasma membrane. The 6 peripheral SNAREpins are coordinately assembled during this transition. The 6 central SNAREpins are assembled later, each by one subunit of the Munc13 hexagon as it flattens onto the plasma membrane to form an outer ring. The central SNAREpins are bound to the inner Ca++-sensitive ring of Synaptotagmin, each paired with a peripheral SNAREpin by a bridging molecule of Complexin in the previously observed trans-clamping arrangement. Binding of Ca++ then releases all 12 SNAREpins simultaneously. This model makes many novel and testable predictions and is consistent with available structural and genetic evidence.

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Monday Speaker Abstracts

MEMBRANE FUSION AND FISSION: LESSONS FROM UNEXPECTED PLACES

Erdem Karatekin ; 1 Yale University, Cellular and Molecular Physiology, New Haven, CT, USA

In mammalian cells specialized for secretion, why do some secretory vesicles collapse rapidly into the plasma membrane after fusing with it, while some others undergo reuptake nearly intact? In bacteria, how does membrane fission occur during endospore formation? I will show evidence that in both cases cell membrane flows, driven by membrane tension gradients, seems to be critical.

MOLECULAR REGULATION OF ATRIAL NATRIURETIC PEPTIDE SECRETION IN CARDIOMYOCYTES BY THE RAB3GAP1 AND THE RAB3A GTPASE CYCLE

Kobina Essandoh 1 ; Arasakumar Subramani 1 ; James P Teuber 1 ; Matthew J Brody 1,2 ; 1 University Of Michigan, Pharmacology, Ann Arbor, MI, USA 2 University of Michigan, Internal Medicine, Division of Cardiovascular Medicine, Ann Arbor, MI, USA

Despite the clinical relevance to cardiovascular disease, molecular regulation of atrial natriuretic peptide (ANP) release by cardiomyocytes remains ill-defined. We uncovered novel functions for Rab3gap1 and modulation of Rab3a nucleotide cycling in membrane fusion and atrial natriuretic peptide (ANP) release. We previously generated transgenic mice overexpressing the Golgi localized S-acyltransferase, zDHHC9, and identified Rab3gap1 as a novel cardiac substrate of zDHHC9. Rab3gap1 palmitoylation and retention at the cardiomyocyte Golgi, Rab3a-GTP levels, and formation of Rab3a-positive post-Golgi vesicles were all enhanced in zDHHC9 overexpressing hearts as were Rab3gap1, Rab3a, and ANP atrial protein levels but circulating ANP levels were reduced, suggesting a defect in ANP secretion. Notably, treatment of cardiomyocytes with the secretagogue phenylephrine (PE) promoted substantial ANP release as expected, but also enhanced Rab3gap1 palmitoylation and Rab3a GTP-loading and peripheral vesicle formation, whereas knockdown of zDHHC9 repressed these PE-induced effects on Rab3gap1 and Rab3a but further enhanced PE-stimulated ANP secretion, suggesting zDHHC9 mediated palmitoylation of Rab3gap1 serves as an intrinsic mechanism to limit ANP release. Consistent with a role for Rab3a nucleotide exchange in modulation of ANP release, overexpression of Rab3gap1 in cardiomyocytes promoted a profound increase in ANP release whereas a GAP-deficient mutant of Rab3gap1 (Rab3gap1 R728A ) was resistant to this effect. Collectively, these data suggest disruption of Rab3a nucleotide cycling perturbs secretory vesicle membrane fusion and ANP release in cardiac myocytes and establish critical roles for Rab3a activity and its modulation by zDHHC9-mediated palmitoylation of Rab3gap1 in governing cardiac homeostasis and endocrine signaling. Future studies in mice with cardiomyocyte-specific deletion of Rab3gap1 and zDHHC9 will further examine the necessity of Rab3a GTP:GDP exchange on ANP secretion and blood pressure homeostasis in vivo.

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Monday Speaker Abstracts

SIGNALING NETWORKS COUPLING CALCIUM TO SECRETION IN THE ADRENAL MEDULLA

Arun Anantharam 1 ; 1 University of Toledo, Neurosciences, Toledo, OH, USA

The adrenomedullary chromaffin cell is an important effector of the autonomic nervous system in the periphery. It responds to sympathetic stimulation by releasing hormones—including the catecholamines, epinephrine and norepinephrine—into the circulation. The sympathetic, “splanchnic” fibers that innervate chromaffin cells house a variety of peptide neurotransmitters which are thought to be co-released with acetylcholine onto the adrenal medulla. However, unlike the case with acetylcholine, the effects of peptidergic stimulation on catecholamine release remain poorly understood. The focus of this talk is on a particular peptide neurotransmitter – pituitary adenylate cyclase activating polypeptide (PACAP) – which has recently emerged as a “stress transmitter” at the splanchnic-chromaffin cell synapse. Here, the intracellular mechanisms that underlie the effects of PACAP on chromaffin cell secretion will be discussed. A model, buttressed by imaging, electrophysiological, and pharmacological data, is proposed, wherein PACAP, through a cascade of effectors, drives secretion. A key hub in this effector network is a phospholipase C epsilon (PLCe) protein, without which neither increases in intracellular calcium, nor secretion, are observed. Overall, the talk will provide new molecular insights on how peptide signals are transduced into adrenomedullary outputs, especially during the stress response.

NEGATIVE REGULATION OF EXOCYTOSIS BY AMISYN Ira Milosevic 1,2 ; 1 University of Oxford , Nuffield Department of Medicine, Oxford, United Kingdom 2 Multidisciplinary Institute for Ageing, Coimbra, Portugal No Abstract

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Monday Speaker Abstracts

RECONSTITUTING ESCRT-III MEDIATED MEMBRANE FISSION IN VITRO

Aurelien Roux 1 ; Javier Espadas 1 ; Anna Pfitzner 1 ; Cesar Bernat 1 ; Henry Zivkovitch 1 ; 1 University of Geneva, Biochemistry, Geneva, Switzerland

The ESCRT-III complex is the only fission machinery known to break lipid membranes from within the neck, in an orientation inverse to dynamin. It is also one of the most ancient membrane remodelling machinery, the only one present in old Archaea species. In the recent years, we have discovered essential features of the ESCRT-III that explain how it deforms the membrane. But the high dynamics of the fission reaction is difficult to reconstitute in vitro. By using Archeal proteins, I will present recent data towards the reconstitution of ESCRT-III mediated membrane fission.

AUTOPHAGY-LYSOSOMAL PATHWAYS IN NEURONS AND ASTROCYTES

Sandra Maday 1 ; David K Sidibe 1 ; Max H Stempel 1 ; Maeve L Coughlan 1 ; Christina Miranda 1 ; 1 Perelman School of Medicine at the University of Pennsylvania, Department of Neuroscience, Philadelphia, PA, USA

Neurons and astrocytes collaborate to construct trillions of synaptic connections in the brain. Most neurons and astrocytes are post-mitotic and long-lived, requiring robust quality control pathways to regulate the integrity of the proteome. Moreover, neurons and astrocytes have cell type-specific functions that place unique demands on the proteome. A key regulator of the proteome is autophagy, a lysosomal degradation pathway. During autophagy, cellular components are packaged into autophagosomes which fuse with lysosomes to enable cargo degradation. Loss of autophagy causes neurodevelopmental defects and neurodegeneration in mice and humans. How autophagy is regulated in neurons and astrocytes to facilitate cell-type specific functions and responses to cellular stress is largely unknown. Here, I present our work on defining pathways for autophagy in neurons and astrocytes. We developed a robust system to coculture primary neurons and astrocytes that recapitulates morphological, proteomic, and functional signatures of astrocytes in vivo. We find that the autophagy receptor p62 and its substrate engagement are differentially regulated in neurons and astrocytes. Metabolic stress increases p62-positive puncta in neurons and astrocytes, but only in neurons do these p62 puncta robustly associate with ubiquitin and require ubiquitination for their formation. These differences were also revealed at baseline. Moreover, deletion of the ubiquitin-associated (UBA) domain reduced p62 puncta only in neurons. In astrocytes, deletion of the UBA domain reduced p62 localization to aggresomes with proteasome-inhibition. Thus, our data suggest that neurons and astrocytes manage quality control differently and p62 has cell-type-specific functions that are elicited in distinct paradigms of cellular stress. Since ALS-linked mutations fall within distinct functional domains of p62, our study provides insights into cell-type-specific contributions to ALS. In fact, ALS-linked mutations in p62 that impair ubiquitin-binding disrupt baseline selective autophagy preferentially in neurons. Our work sets the stage for defining intercellular pathways between neurons and astrocytes to regulate the proteome.

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Membrane Budding and Fusion

Monday Speaker Abstracts

MODEL FOR RING COLLAPSE AND MEMBRANE FISSION IN ER TUBULAR NETWORKS

Michael M. Kozlov ; 1 Tel Aviv University, Tel Aviv, Israel

Tubular networks of the endoplasmic reticulum (ER) are dynamic structures whose steady-state conformations are maintained by a balance between the persistent generation and vanishing of the network elements. While factors producing the ER tubules and inter-tubular junctions have been investigated, the mechanisms behind their elimination remained unknown. Here we addressed the ER ring closure, the process resulting in the tubule and junction removal through constriction of the network unit-cells into junctional knots followed by the knot remodeling into regular junctions. We considered the ring closure to be driven by the tension existing in ER membranes. We modeled, computationally, the structures of the junctional knots containing internal nanopores and analyzed their tension dependence. We predicted an effective interaction between the nanopores facilitating the knot tightening and collapse of additional network unit cells. We analyzed the process of the pore sealing through membrane fission resulting in formation of regular junctions. Considering the hemi-fission as the rate-limiting stage of the fission reaction, we evaluated the membrane tensions guarantying the spontaneous character of the pore sealing. We concluded that feasible membrane tensions explain all stages of the ER ring closure.

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Membrane Budding and Fusion

Monday Speaker Abstracts

RECONSTRUCTING THE COLLECTIVE BEHAVIOUR OF THE ENDOSOMAL MEMBRANE FUSION MACHINERY Lisa Redlingshöfer 1,2 ; Mareike Jordan 1 ; Swantje Lenz 1 ; Alexander von Appen 1 ; Stephan Grill 1,2 ; Marino Zerial 1 ; 1 Max Planck Institute of Molecular Cell Biology and Genetics, Dresden, Germany 2 Technische Universität Dresden, Dresden, Germany The Rab5 GTPase determines the location and timing of endosomal membrane fusion. In order to do so, Rab5 is activated on early endosomal membranes by the Rabex5:Rabaptin5 complex, which enables the recruitment of a plethora of Rab5 effector proteins and the formation of high molecular weight oligomers. Yet, in the absence of PI3P, the hallmark phosphoinositide lipid of the early endosome, Rab5 is unable to recruit the full fusion machinery. To understand how the interplay of protein-protein and protein-lipid interactions creates a fully functional collective, we probed the assembly dynamics and characterised the structural arrangement of the endosomal fusion machinery. To this end, we combined in vitro reconstitution experiments on liposomes and supported lipid bilayers with cryoEM, crosslinking mass spectrometry and TIRF microscopy analysis. Our investigations revealed a novel binding interaction of Rabex5:Rabaptin5 with PI3P, which could serve to target Rab5 to early endosomes. Varying the concentration of PI3P or combination of effector proteins affected assembly dynamics and morphology. Furthermore, we found that Rabex5:Rabaptin5 displays significant conformational variability, which is likely to affect its ability to bind to PI3P and other Rab5 effector proteins. Our findings suggest that PI3P assumes a critical role in mediating the protein-protein interactions necessary to assemble the full Rab5-mediated membrane fusion machinery.

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Membrane Budding and Fusion

Monday Speaker Abstracts

FUNCTION OF ABC G1/G4 TRANSPORTERS AND CHOLESTEROL IN CELL-CELL FUSION

Elizabeth Chen ; 1 UT Southwestern Medical Center, Dallas, TX, USA

Cell-cell fusion is critical for the development and physiology of multicellular organisms. It is an asymmetric process in which an invading cell drills actin-propelled membrane protrusions into the receiving cell to promote cell membrane juxtaposition and fusion. Although the protein machinery at the site of fusion, known as the fusogenic synapse, has been extensively studied, the function of lipids in the fusion process remains largely unknown. In a screen for transmembrane proteins involved in cell-cell fusion, we identified a function for the G1/G4 family of ATP-binding cassette transporters (ABC transporters). Overexpression of ABCG1/4 transporters significantly enhanced cell-cell fusion, accompanied by the enrichment of free cholesterol on the outer leaflet of the plasma membrane. In contrast, extracting free cholesterol from the plasma membrane by treating cells with β -methylcyclodextrin inhibited cell-cell fusion, demonstrating a critical function for cholesterol in cell-cell fusion. Using TIRF microscopy, we observed enrichment of free cholesterol associated with the invasive membrane protrusions at the fusogenic synapse. Consistent with this, overexpression of ABCG1/4 transporters in Drosophila S2R+ cells led to an overall increase in the number of membrane protrusions. In contrast, knocking down ABCG1/4 transporters in mouse myoblasts inhibited cell-cell fusion, accompanied by less free cholesterol on the plasma membrane and decreased number of protrusions. Using probes for membrane fluidity and disordered lipid phase, we show that free cholesterol on the outer leaflet significantly increased membrane fluidity and disordered domains. Taken together, our studies revealed a novel function for ABCG1/4 transporters in cell cell fusion and uncovered a critical role for free cholesterol in the fusion process. We further demonstrate that free cholesterol enhances membrane fluidity and thus allowing the formation of membrane protrusions required for cell-cell fusion.

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Membrane Budding and Fusion

Monday Speaker Abstracts

MECHANISTIC ANALYSIS OF MEMBRANE FISSION AND DISCOVERY OF NOVEL FISSION PROTEINS

Thomas J. Pucadyil ; 1 Indian Institute of Science Education and Research, Pune, Biology, Pune, India

The lipid bilayer is highly resilient to rupture and explains why it was selected over the course of evolution to serve a barrier function. Yet fission, or the splitting of a membrane compartment, is a central theme in biology that manifests during cell division, organelle biogenesis and vesicular transport. Fission involves the local application of forces to bend and constrict a tubular membrane intermediate. Using a facile assay system of supported membrane nanotubes that can be tuned for size and lipid composition, we have analyzed fission mechanisms of candidate proteins and discovered novel proteins that catalyze fission. My talk will describe recent developments in our efforts at understanding the pathway to fission and expanding the repertoire of fission proteins.

HARNESSING ACTIN ASSEMBLY FORCES TO DRIVE VESICLE FORMATION DURING CLATHRIN-MEDIATED ENDOCYTOSIS

David G. Drubin ; Daniel G Serwas 1 ; Jennifer G Hill 1 ; Matt Akamatsu 1 ; Ross Pedersen 1 ; 1 UC Berkeley, Molecular and Cell Biology, Berkeley, CA, USA

From yeast to humans, forces generated by the actin cytoskeleton assist in formation of clathrin coated vesicles during endocytosis. While the principles for how actin assembly generates pushing forces for cell motility have been well established, how actin assembly generates pulling forces for vesicle formation is not well understood. Studies conducted examined the ultrastructure of the actin cytoskeleton at endocytic sites, and investigated how cross-linking proteins, myosins and a force-sensitive coat protein, Hip1R/Sla2, generate pulling forces. Single molecule biophysics shows that the myosin is an active motor that generates pulling force. Actin assembly in mammalian cells is asymmetric at endocytic sites, while in yeast assembly occurs uniformly around the endocytic site. When membrane tension increases, load adaptation mechanisms increase actin assembly, suggesting coupling between force-sensing proteins and the actin assembly machinery. An integrated model for how actin mediates endocytic vesicle formation will be presented.

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Membrane Budding and Fusion

Monday Speaker Abstracts

SELF-ASSEMBLY AND STRUCTURE OF A CLATHRIN-INDEPENDENT AP-1:ARF1 TUBULAR MEMBRANE COAT

Rick Hooy 1,2 ; Yuichiro Iwamoto 1,2 ; Dan A Tudorica 2,3 ; Xuefeng Ren 1,2 ; James H Hurley 1,2,4 ; 1 University of California, Berkeley, Molecular and Cell Biology, Berkeley, CA, USA 2 California Institute for Quantitative Biosciences, University of California, Berkeley , Berkeley, CA, USA 3 Graduate Group in Biophysics, University of California, Berkeley, Berkeley, CA, USA 4 Helen Wills Neuroscience Institute, University of California, Berkeley, Berkeley, CA, USA

The adaptor protein (AP) complexes not only form the inner layer of clathrin coats but also have clathrin-independent roles in membrane traffic. How APs assemble on membranes to achieve diverse cargo trafficking functions (e.g. with and without clathrin) remains ill defined. Pathogens co-opt AP assemblies to (de)regulate trafficking of select cargo molecules; HIV-1 Nef hijacks AP-1 and its cofactor, Arf1, to sequester major histocompatibility complex class I (MHC-I) to evade immune detection. To determine how Nef hijacks AP-1:Arf1 cargo trafficking assemblies, we reconstituted Nef-mediated MHC-I downregulation in vitro and solved the subnanometer resolution structures of membrane-associated AP-1 assemblies by cryo-electron tomography and subtomogram averaging. We found that AP-1:Arf1:Nef:MHC-I forms a coat on tubulated membranes without clathrin. The coat assembles via Arf1 dimer interfaces. The stoichiometry of AP-1-to-Arf1 correlates with membrane tube diameter. In cells, AP-1–positive tubules are enriched upon clathrin knockdown. Nef localizes preferentially to AP-1 tubules in cells, explaining how Nef sequesters MHC-I. Coat contact residues are conserved across Arf homologs and the Arf-dependent AP complexes AP-1, AP-3, and AP-4. Thus, AP complexes can self assemble with Arf1 into tubular coats without clathrin or other scaffolding factors. The AP 1:Arf1 coat defines the structural basis of a broader class of tubulovesicular membrane coats, as an intermediate in clathrin vesicle formation from internal membranes and as an MHC-I sequestration mechanism in HIV-1 infection.

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Membrane Budding and Fusion

Monday Speaker Abstracts

MOLECULAR MECHANOSENSING IN DYNAMIN-DRIVEN MEMBRANE REMODELLING

Vadim A Frolov 1 ; Javier Vera Lillo 1 ; Pedro Arrasate 1 ; Pavel V Bashkirov 2 ; 1 University of the Basque Country, Biophysics Institute, Leioa, Spain 2 Research Institute for Systems Biology and Medicine, Moscow, Russian Federation

Dynamin 1 GTPase (Dyn1) is a mechano-enzyme involved in membrane fission during synaptic vesicle endocytosis. While the overall structure of the Dyn1 fission machinery, a short helical polymer, is well understood, there is ongoing debate surrounding its operational principles. Although much of the discussion has centered around how the Dyn1 helix, driven by GTP hydrolysis, induces membrane curvature, our findings demonstrate that the GTPase activity of Dyn1 should be considered within the broader context of its integration into the membrane remodeling circuitry at the synapse. Through a series of single-molecule in vitro assays, we reveal that Dyn1 utilizes hydrolysis not only to generate membrane curvature and stress but also for mechanosensitive reassembling. Importantly, the discrimination between membrane curvature and rigidity is already encoded within subhelical Dyn1 oligomers, enabling rapid subsecond membrane fission and facilitating the prompt recycling of Dyn1 machinery for subsequent actions. This capability is crucial for supporting the high turnover of synaptic membrane required for efficient synaptic vesicle recycling.

MITOCHONDRIAL DERIVED VESICLES: A NEW MITOCHONDRIAL QUALITY CONTROL MECHANISM Chantell S. Evans ; 1 Duke University Medical Center, Cell Biology, Durham, NC, USA No Abstract

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Membrane Budding and Fusion

Monday Speaker Abstracts

BIOGENESIS OF RNA-CONTAINING EXTRACELLULAR VESICLES AT ER MEMBRANE CONTACT SITES

Alissa M. Weaver 1 ; 1 Vanderbilt University School of Medicine, Cell and Developmental Biology, Nashville, TN, USA

Extracellular RNA is a novel mechanism for cell-to-cell communication and drives many physiological and pathological processes including cancer. Extracellular vesicles (EVs) are a major vehicle for transmitting these RNAs between cells. However, the underlying mechanisms by which RNA-containing EVs are generated are poorly understood. Due to the association of many RNA processing granules with the endoplasmic reticulum, we investigated the role of endoplasmic reticulum membrane contact sites (ER MCS) as key subcellular locations for the biogenesis of RNA-containing EVs. We found that inhibition or overexpression of key molecules that control ER MCS (VAP-A-KD, VAP-A OE) and ceramide transport at ER MCS (CERT-KD) greatly affected the small RNA content of both small and large EVs. Density gradient sub-fractionation of EV pellets revealed that VAP-A regulates a select subpopulation of small EVs that are enriched with RNA. Confocal microscopy data revealed that key RNA and RNA binding proteins are altered in multivesicular endosomes in VAP-A KD cells. Experiments testing EV function indicated that this VAP-A-controlled small EV population is critical for both in vitro transfer of miR-100 to recipient cells and for in vivo growth of xenograft mouse tumors. Altogether, these and additional data suggest a model in which ceramide transfer at ER MCS drives biogenesis of a select subpopulation of EVs containing RNA-RBP complexes. We are now actively investigating regulatory mechanisms that control this key sorting event, as well as the functional consequences.

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