Plasma membrane damage removal by F-actin-mediated shedding from repurposed filopodia

Repairing plasma membrane damage is vital to eukaryotic cell survival. Membrane shedding is thought to be key to this repair process, but a detailed view of how the process occurs is still missing. Here we used electron cryotomography to image the ultrastructural details of plasma membrane wound healing. We found that filopodia-like protrusions are built at damage sites, accompanied by retraction of neighboring filopodia, and that these repurposed protrusions act as scaffolds for membrane shedding. This suggests a new role for filopodia as reservoirs of membrane and actin for plasma membrane damage repair. Damage-induced shedding was dependent on F-actin dynamics and Myo1a, as well as Vps4B, an important component of the ESCRT machinery. Thus we find that damage shedding is more complex than current models of simple vesiculation from flat membrane domains. Rather, we observe structural similarities between damage-mediated shedding and constitutive shedding from enterocytes that argue for conservation of a general membrane shedding mechanism

Stable sub-complexes observed in situ suggest a modular assembly pathway of the bacterial flagellar motor

The self-assembly of cellular macromolecular machines such as the bacterial flagellar motor requires the spatio- temporal synchronization of gene expression, protein localization and association of a dozen or more unique components. In Salmonella and Escherichia coli, a sequential, outward assembly mechanism has been proposed for the flagellar motor starting from the inner membrane, with each subsequent component stabilizing the last. Here, using electron cryo-tomography of intact Legionella pneumophila, Pseudomonas aeruginosa and Shewanella oneidensis cells, we observe stable outer-membrane-embedded sub-complexes of the flagellar motor. These sub- complexes consist of the periplasmic embellished P- and L-rings, in the absence of other flagellar components, and bend the membrane inward dramatically. Additionally, we also observe independent inner-membrane sub- complexes consisting of the C- and MS-rings and export apparatus. These results suggest an alternate model for flagellar motor assembly in which outer- and inner-membrane-associated sub-complexes form independently and subsequently join, enabling later steps of flagellar production to proceed

In situ imaging of the bacterial flagellar motor disassembly and assembly processes

The self‐assembly of cellular macromolecular machines such as the bacterial flagellar motor requires the spatio‐temporal synchronization of gene expression with proper protein localization and association of dozens of protein components. In Salmonella and Escherichia coli, a sequential, outward assembly mechanism has been proposed for the flagellar motor starting from the inner membrane, with the addition of each new component stabilizing the previous one. However, very little is known about flagellar disassembly. Here, using electron cryo‐tomography and sub‐tomogram averaging of intact Legionella pneumophila, Pseudomonas aeruginosa, and Shewanella oneidensis cells, we study flagellar motor disassembly and assembly in situ. We first show that motor disassembly results in stable outer membrane‐embedded sub‐complexes. These sub‐complexes consist of the periplasmic embellished P‐ and L‐rings, and bend the membrane inward while it remains apparently sealed. Additionally, we also observe various intermediates of the assembly process including an inner‐membrane sub‐complex consisting of the C‐ring, MS‐ring, and export apparatus. Finally, we show that the L‐ring is responsible for reshaping the outer membrane, a crucial step in the flagellar assembly process

INS1-E cells exp Mito-DsRed

No correlations done. Just a survey of these secretory cells.<strong>Tilt Series Date:</strong> 2016-07-23</p> <strong>Data Taken By:</strong> Shrawan kumar Mageswaran</p> <strong>Species / Specimen:</strong> Unspecified</p> <strong>Strain:</strong> None</p> <strong>Tilt Series Settings:</strong> Single Axis, tilt range: (-55.0°, 55.0°), step: 1°, constant angular increment, dosage: -50.0 eV/Ų, defocus: -8.0 μm, magnification: 18000x. </p> <strong>Microscope:</strong> Caltech Polara</p> <strong>Acquisition Software:</strong> SerialEM</p> <strong>Upload Method:</strong> reupload_downloaded</p> <strong>Processing Software Used:</strong> IMOD</p> <strong>Collaborators and Roles:</strong> Stephen's collaborator Zachary Freyberg at U.Pitt cultured, transfected and froze cells.</p>Files available via S3 at https://renc.osn.xsede.org/ini210004tommorrell/tomography_archive/smk2016-07-23-23</p>INS1E-mitoDR-surveyTS22.mrc, Tilt Series (Pixel Size 0.6 nm), 1.7 GB <a role="button" class="ui compact mini button" href="https://renc.osn.xsede.org/ini210004tommorrell/tomography_archive/smk2016-07-23-23/rawdata/INS1E-mitoDR-surveyTS22.mrc" > <i class="download icon"></i> Download </a></p> INS1E-mitoDR-surveyTS22.rec, Reconstruction (Pixel Size 2.4 nm), 2.1 GB <a role="button" class="ui compact mini button" href="https://renc.osn.xsede.org/ini210004tommorrell/tomography_archive/smk2016-07-23-23/3dimage_79041/INS1E-mitoDR-surveyTS22.rec" > <i class="download icon"></i> Download </a></p> INS1E-mitoDR-surveyTS22_DB.rec, Reconstruction (Pixel Size 0.0 nm), 341.4 MB <a role="button" class="ui compact mini button" href="https://renc.osn.xsede.org/ini210004tommorrell/tomography_archive/smk2016-07-23-23/3dimage_81498/INS1E-mitoDR-surveyTS22_DB.rec" > <i class="download icon"></i> Download </a></p&gt

Saccharomyces cerevisiae - JAWY1

Raw data files of Saccharomyces cerevisiae - JAWY1<strong>Tilt Series Date:</strong> 2016-11-30</p> <strong>Data Taken By:</strong> Shrawan kumar Mageswaran</p> <strong>Species / Specimen:</strong> Unspecified</p> <strong>Strain:</strong> None</p> <strong>Tilt Series Settings:</strong> Single Axis, tilt range: (-60.0°, 60.0°), step: 2°, constant angular increment, dosage: 150.0 eV/Ų, defocus: -6.0 μm, magnification: 27500x. </p> <strong>Microscope:</strong> Caltech Polara</p> <strong>Acquisition Software:</strong> SerialEM</p> <strong>Upload Method:</strong> pipeline</p> <strong>Processing Software Used:</strong> IMOD</p> <strong>Collaborators and Roles:</strong> Obtained from Dr. Greg Odorizzi @ Univ of Colorado at Boulder.</p> <strong>Purification / Growth Conditions / Treatment:</strong> Cells were grown overnight, diluted to 0.2 OD in the morning and allowed to grow till OD ~0.6. sm2016-11-30-1to5,*-10,*-13to20 (TS1-TS14)- Heat shocked @38 deg C for 2min10min. sm2016-11-30-6to9,*-11to12 (TS15-21) - Heat shocked @38 deg C for 1.5 hr and recovered @28deg C for 40 min.</p>Files available via S3 at https://renc.osn.xsede.org/ini210004tommorrell/tomography_archive/smk2016-11-30-18</p>TS7.mrc, Tilt Series (Pixel Size 0.39 nm), 1.5 GB <a role="button" class="ui compact mini button" href="https://renc.osn.xsede.org/ini210004tommorrell/tomography_archive/smk2016-11-30-18/rawdata/TS7.mrc" > <i class="download icon"></i> Download </a></p&gt

COS-7 expressing HIV-1 Gag

Raw data files of COS-7 expressing HIV-1 Gag<strong>Tilt Series Date:</strong> 2015-08-06</p> <strong>Data Taken By:</strong> Shrawan kumar Mageswaran</p> <strong>Species / Specimen:</strong> Cos7</p> <strong>Strain:</strong> Cos7 cells</p> <strong>Tilt Series Settings:</strong> Single Axis, tilt range: (-60.0°, 60.0°), step: 1°, constant angular increment, dosage: 80.0 eV/Ų, defocus: -8.0 μm, magnification: 18000x. </p> <strong>Acquisition Software:</strong> UCSFTomo</p> <strong>Upload Method:</strong> pipeline</p> <strong>Processing Software Used:</strong> IMOD</p>Files available via S3 at https://renc.osn.xsede.org/ini210004tommorrell/tomography_archive/smk2015-08-06-24</p>GDgrid_atlas1_HIV_COS7par0020_full.rec, Reconstruction (Pixel Size 2.4 nm), 445.4 MB <a role="button" class="ui compact mini button" href="https://renc.osn.xsede.org/ini210004tommorrell/tomography_archive/smk2015-08-06-24/3dimage_60189/GDgrid_atlas1_HIV_COS7par0020_full.rec" > <i class="download icon"></i> Download </a></p> GDgrid_atlas1_HIV_COS7par0020.mrc, Tilt Series (Pixel Size 0.6 nm), 3.4 GB <a role="button" class="ui compact mini button" href="https://renc.osn.xsede.org/ini210004tommorrell/tomography_archive/smk2015-08-06-24/rawdata/GDgrid_atlas1_HIV_COS7par0020.mrc" > <i class="download icon"></i> Download </a></p&gt

COS-7 expressing HIV-1 Gag

Raw data files of COS-7 expressing HIV-1 Gag<strong>Tilt Series Date:</strong> 2015-08-06</p> <strong>Data Taken By:</strong> Shrawan kumar Mageswaran</p> <strong>Species / Specimen:</strong> Cos7</p> <strong>Strain:</strong> Cos7 cells</p> <strong>Tilt Series Settings:</strong> Single Axis, tilt range: (-60.0°, 60.0°), step: 1°, constant angular increment, dosage: 80.0 eV/Ų, defocus: -8.0 μm, magnification: 18000x. </p> <strong>Acquisition Software:</strong> UCSFTomo</p> <strong>Upload Method:</strong> pipeline</p> <strong>Processing Software Used:</strong> IMOD</p>Files available via S3 at https://renc.osn.xsede.org/ini210004tommorrell/tomography_archive/smk2015-08-06-29</p>GDgrid_atlas1_HIV_COS7par0025_full.rec, Reconstruction (Pixel Size 2.4 nm), 445.4 MB <a role="button" class="ui compact mini button" href="https://renc.osn.xsede.org/ini210004tommorrell/tomography_archive/smk2015-08-06-29/3dimage_60197/GDgrid_atlas1_HIV_COS7par0025_full.rec" > <i class="download icon"></i> Download </a></p> GDgrid_atlas1_HIV_COS7par0025.mrc, Tilt Series (Pixel Size 0.6 nm), 3.4 GB <a role="button" class="ui compact mini button" href="https://renc.osn.xsede.org/ini210004tommorrell/tomography_archive/smk2015-08-06-29/rawdata/GDgrid_atlas1_HIV_COS7par0025.mrc" > <i class="download icon"></i> Download </a></p&gt

Yeast 6210 - Nup133delC

To study clustering phenotype of nuclear pore complexes. SEY6210 transformed with Nup133dC + Nup49-GFP (Leu)<strong>Tilt Series Date:</strong> 2018-11-27</p> <strong>Data Taken By:</strong> Shrawan kumar Mageswaran</p> <strong>Tilt Series Settings:</strong> Single Axis, tilt range: (-60.0°, 60.0°), step: 2°, constant angular increment, dosage: 100.0 eV/Ų, defocus: -8.0 μm, magnification: 33000x. </p> <strong>Microscope:</strong> Caltech Titan Krios</p> <strong>Acquisition Software:</strong> SerialEM</p> <strong>Upload Method:</strong> reupload_downloaded</p> <strong>Processing Software Used:</strong> IMOD, tomo3D</p> <strong>Collaborators and Roles:</strong> Karsten from Hoelz lab made the strain.</p> <strong>Purification / Growth Conditions / Treatment:</strong> (Hoeltz Lab) Cells were grown overnight in -Leu medium to 0.68 OD (a bit slow growing) @30 C. Cells were diluted to 0.25 OD for freezing in the same medium. Cells were kept on ice (for ~2 hr) until freezing.</p> <strong>Sample Preparation:</strong> Glow discharged Cu finder grids. Added 3.5 µL of cell suspension and blotted for ~5 sec from behind. temp 6C, RH 100%. FIB-milled.</p>Files available via S3 at https://renc.osn.xsede.org/ini210004tommorrell/tomography_archive/smk2018-11-27-21</p>TS13-pt65.mrc, Tilt Series (Pixel Size 0.432 nm), 1.7 GB <a role="button" class="ui compact mini button" href="https://renc.osn.xsede.org/ini210004tommorrell/tomography_archive/smk2018-11-27-21/rawdata/TS13-pt65.mrc" > <i class="download icon"></i> Download </a></p> TS13_4bin_SIRTl20_rotx.rec, Reconstruction (Pixel Size 0.0 nm), 1.6 GB <a role="button" class="ui compact mini button" href="https://renc.osn.xsede.org/ini210004tommorrell/tomography_archive/smk2018-11-27-21/3dimage_102054/TS13_4bin_SIRTl20_rotx.rec" > <i class="download icon"></i> Download </a></p&gt

INS1-E cells exp Mito-DsRed

No correlations done. Just a survey of these secretory cells.<strong>Tilt Series Date:</strong> 2016-07-23</p> <strong>Data Taken By:</strong> Shrawan kumar Mageswaran</p> <strong>Species / Specimen:</strong> Unspecified</p> <strong>Strain:</strong> None</p> <strong>Tilt Series Settings:</strong> Single Axis, tilt range: (-55.0°, 55.0°), step: 1°, constant angular increment, dosage: -50.0 eV/Ų, defocus: -8.0 μm, magnification: 18000x. </p> <strong>Microscope:</strong> Caltech Polara</p> <strong>Acquisition Software:</strong> SerialEM</p> <strong>Upload Method:</strong> reupload_downloaded</p> <strong>Processing Software Used:</strong> IMOD</p> <strong>Collaborators and Roles:</strong> Stephen's collaborator Zachary Freyberg at U.Pitt cultured, transfected and froze cells.</p>Files available via S3 at https://renc.osn.xsede.org/ini210004tommorrell/tomography_archive/smk2016-07-23-21</p>INS1E-mitoDR-surveyTS20.mrc, Tilt Series (Pixel Size 0.6 nm), 1.7 GB <a role="button" class="ui compact mini button" href="https://renc.osn.xsede.org/ini210004tommorrell/tomography_archive/smk2016-07-23-21/rawdata/INS1E-mitoDR-surveyTS20.mrc" > <i class="download icon"></i> Download </a></p> INS1E-mitoDR-surveyTS20_part61_5.rec, Reconstruction (Pixel Size 2.4 nm), 1.1 GB <a role="button" class="ui compact mini button" href="https://renc.osn.xsede.org/ini210004tommorrell/tomography_archive/smk2016-07-23-21/3dimage_79036/INS1E-mitoDR-surveyTS20_part61_5.rec" > <i class="download icon"></i> Download </a></p> INS1E-mitoDR-surveyTS20_DB.rec, Reconstruction (Pixel Size 0.0 nm), 332.5 MB <a role="button" class="ui compact mini button" href="https://renc.osn.xsede.org/ini210004tommorrell/tomography_archive/smk2016-07-23-21/3dimage_81496/INS1E-mitoDR-surveyTS20_DB.rec" > <i class="download icon"></i> Download </a></p&gt