MSBS 5065S: Cellular Structure and Function Lecture 1 PDF

MSBS 5065S: Cellular Structure and Function Joe Kramer, Ph.D. (Dept. of Pathology) Jia Shi, Ph.D. (Dept. of Pharmacology) Course Introduction/Microscopy 9_6_2023  MSBS 5065S: Cellular Structure and Function Class: Tuesdays, 12:10-1:30 and Fridays, 10:20-11:40 Offic...

MSBS 5065S: Cellular Structure and Function Joe Kramer, Ph.D. (Dept. of Pathology) Jia Shi, Ph.D. (Dept. of Pharmacology) Course Introduction/Microscopy 9_6_2023  MSBS 5065S: Cellular Structure and Function Class: Tuesdays, 12:10-1:30 and Fridays, 10:20-11:40 Office Hours: Dr. Kramer; Fridays 1:00 (subject to change) Exams: October 11 (4-6PM); November 19(6-8PM); December 23(2-4PM) TBLs: 9/26; 12/12 -- 6:00-7:30 Review Sessions: See schedule Optional Textbook: Molecular Biology of the Cell 7th edition, https://wwnorton.com/books/9780393884821 Other Materials: Occasionally, papers will be assigned to augment coverage of selected topics. These materials will be available on the course canvas site.  Course Information We are using Canvas Learning management system https://canvas.rutgers.edu You should have been invited to the Cell Course Site CSF Canvas Site If not, please let me know as soon as possible. The Canvas site contains: Slides, schedule, course information, lecture recordings, grades interface, ect. Accessing Slides and Video Podcasts Canvas Menu Topics have their own modules and will have a pdf of the slides for each lecture as well as a link to the video recording Lecture recordings and video items will be found here and in Module associated with specific lecture Lecture 6-SepIntroduction -- Microscopy and Basic Methodolgies 10-SepNuclear Structure and Protein Import East Lecture Hall East Lecture Hall Kramer Kramer Schedule 13-SepChromatin Structure and Epigenetics 17-SepMembrane Biochemistry 20-SepMembrane trafficking -- Vesicular reactions 24-SepMembrane trafficking -- Vesicular Trafficking East Lecture Hall East Lecture Hall Main Lecture Hall East Lecture Hall Kramer Shi Kramer Kramer 26-SepTeam Based learning #1 Main Lecture Hall Kramer 27-SepCell Signaling I -- Basic Principles and Agents Main Lecture Hall Shi 1-OctCell Signaling II -- G protein Receptors East Lecture Hall Shi 4-OctCell Signaling III -- Receptor Tyrosine Kinases Main Lecture Hall Shi 8-Octpre-Exam Review East Lecture Hall Kramer/Shi 11-OctExam I Main Lecture Hall 15-OctNo class n/a 18-OctCytoskeletal Systems - polymer dynamics TBD Winkelmann 22-OctCytoskeleton II East Lecture Hall Winkelmann 25-OctCytoskeleton III Main Lecture Hall Winkelmann 29-OctCell adhesion I East Lecture Hall Kramer 1-NovCell adhesion II Main Lecture Hall Kramer 5-NovExtracellular matrix/ Mechanotransduction East Lecture Hall Kramer 8-NovCell Motility I East Lecture Hall Kramer 12-NovNo Class n/a 15-NovCell Motility II East Lecture Hall Kramer 18-Novpre-Exam Review TBD 19-NovExam II Main Lecture Hall 22-NovCell Cycle -- CDKs TBD Kramer 26-NovCell Cycle II -- Checkpoints East Lecture Hall Kramer 29-NovNo class meeting- recorded lecture -- Apoptosis ONLINE 3-DecCancer Cell Biology East Lecture Hall Shi 6-DecCancer Cell Biology-Basic principles East Lecture Hall Shi 10-DecCancer Cell Biology -- Pathways East Lecture Hall Shi 12-DecTeam Based learning #2 Main Lecture Hall Kramer/Shi 13-DecStem Cells East Lecture Hall Shi 17-DecNo class n/a 20-Decpre-Exam Review TBD Kramer/Shi 23-DecExam III Main Lecture Hall  Lectures PDFs for the slides presented at the lecture will appear on Canvas by the morning of the scheduled lecture. Documents from other sources (e.g., scientific papers, video links ect.) will be posted. Some will be required and others will be supplemental in nature. Suggested reading material will be designated in the lecture slides posted on Canvas. Attendance is recommended though not required or recorded. Exams and Quizzes A mock quiz will be sent to you to learn how to take a quiz on Examsoft. Three exams, E1(25%), E2 (25%), E3(25%). 3 quizzes equaling 15% (dates to be announced). 2 TBLs equaling 10% Exams and quizzes are a combination of multiple-choice, short answer Will include material discussed in class (slides, papers), textbook readings, and assigned papers. Practice questions/study guides will be made available before exams.  Grading Policy Course grade derives from exams, quizzes, & TBLs entirely. Any excused quizzes/exams must be made-up. Final grades are adjusted to yield a historical distribution of 25-35% A; 35% B+; 35% B; ~5%700nm) (See relationship in Abbe NA=numerical aperture of objective lens equation) = n sin Θ n = refractive index of media Θ = semi-angle of the objective lens Electron beams have a wavelength of picometers thus explaining the ability of Electron Note: A lower value for Microscopes to provide much better resolution resolution is desirable!! than light microscopes.  There is a difference between visualizing and resolving. Resolution of Resolution of Electron Light Microscope microscope = = ~ 10nm 200nm Electron Micrograph Fluorescent Micrograph The microtubules visualized here are 25 nm or so in diameter. This is below the diffraction limited resolution for fluorescent microscopy. Thus in B we can’t definitively say if it is a single microtubule or a bundle of microtubules but we can do so in A.  Why light microscopy is limited… Optical resolutionThe vs. Molecular Diffraction Dimensions Limit Airyspread microscope point Disc function Resolution vs. Molecular Dimensions GFP molecule NA =1.4 !o = 500nm GFP 2.5 nm 2.5 nm 100 nm Even one molecule appears as a broad (>200 nm) point of fluorescence because we are diffraction limited. You cannot know whether this represents 2 or more molecules Super-resolution microscopy Imaging Methods 1 nm SR fluorescence 1 µm microscopy Depth 1 mm 1m 1 nm 1m Lateral Dimension Tsien, Nat. Rev. (2003)  Photoactivatable Fluorescent Proteins – essentialHighlighting for PALM Superres Microscopy using Photoactivation Numerous PA-GFP photoactivatable Photoactivatable GFP Normalized Absorbance proteins have been Photoactivate Preactivation engineered. Post-activation They allow for many useful experiments looking at the dynamics of specific Wavelength (nm) molecules. 488 nm 488 nm They can be turned ~400 nm ON with a focused laser of a specific Pre-activation Post-activation wavelength and the turned OFF by intense laser With respect to SUPER resolution selectively turn on single (photobleached) molecules, image, turn off that molecule and do so again until a super resolution image is obtained.  The Diffraction Limit microscope Airy point Disc spread function NA =1.4 !o = 500nm PAGFP 2.5 nm 100 nm Even one molecule appears as a broad (>200 nm) point of fluorescence because we are diffraction limited. You cannot know whether this represents 2 or more molecules. Unless you image them sequentially by turning the fluorescence ON and OFF PALM Microscopy – PhotoActivatable localization Microscopy From Zeiss website PALM image of Lysosome In practice, current iterations of PALM technology enable ~20 nm resolution.  Diffraction limited Resolution: The Raleigh Criterion defines the lower limit of resolution Due to the wave nature of light an infinitely small point source of light appears as an Airy disc with a number of concentric circles. For two points to be considered distinct the max of one disc must be further than the first dark interference ring of the other = Rayleigh Criterion. The size of the disc, its interference rings and the overall resolution of the optical system is a function of the following: Resolution = 0.61 (λ)/NA λ = wavelength NA=numerical aperture = n sin Θ n = refractive index of media Θ = semi-angle of the objective lens Airy Disc unresolved resolved Until recently, the best optical microscopes have had a resolution of ~ 0.2 μM or 200 nm. This is Note: A lower value for resolution is referred to as the diffraction limited resolution. desirable!!  PALM microscopy – Photo Activation Localization Microscopy Photoactivated – Super-resolution Localization Microscopy (PAL Photoactivate 1st subpopulation Image & then Fit. bleach 2D Plot molecules subpopulation Gaussian. Photoactivate 2nd subpopulation Fit 2D.. Image & then bleach Plot molecules Gaussian subpopulation.. Photoactivate 3rd subpopulation Fit... Image & then bleach 2D Plot molecules subpopulation Gaussian... GFP technology represents a huge advance in our ability to study the in vivo kinetics of biological processes