Unformatted text preview: 2/4/16 CH281 Cells and Organelles / Tissue Engineering Chapter 5 In this lecture… • Importance of cells in the organizaAon of complex biological systems: -‐ ImplicaAons for Assue engineering • Microscopy • Cellular compartments • Organelle structure and funcAon -‐ Secretory pathway compartment -‐ Non-‐secretory compartment 1 2/4/16 Microscopy • History – from the chapter • Microscope types – review chapter examples • Electron microscopy – image created by a paMern of electrons hiNng a detector; image acquired in a vacuum; no living samples ü Transmission electron microscopy -‐ involves chemical stabilizaAon of biological Assue (ﬁxaAon – with aldehydes!), staining with heavy, electron dense metals (osmium, lead, uranium – check the atomic structure), and imaging a very thin sheet of Assue (60 nm) by shooAng a beam of electrons through the Assue ü Scanning electron microscopy – requires ﬁxaAon; coaAng of the outside of a sample with a thin sheet of metal; electrons bounce oﬀ of the surface; detector create image from reﬂected electrons Transmission electron microscopy J Cell Biol. 1998 Jan 12;140(1):39-‐47. 2 2/4/16 Scanning electron microscopy Dartmouth College Electron Microscope Facility Microscopy (cont.) • Light microscopy – image created by the interacAon of light (UV, Visible, IR) with samples chapter ü Brighfield w/stain – light passes through samples; dye used to diﬀerenAate features in the sample (e.g., hematoxylin and eosin stain DNA and protein, respecAvely; “H&E stain”) ü Brighfield w/opAcs – light passes through samples and opAcal methods are used to create contrast based on the diﬀering light paths (phase, diﬀerenAal interference, Nomarski, darkﬁeld, polarizaAon) ü Fluorescence – a stain is selecAvely introduced which is able to absorb energy at one wavelength and emit at another (low background) 3 2/4/16 Histological stain PLoS ONE 4(12): e8157 DiﬀerenAal interference contrast Fluorescence Cancer Res 2006; 66: (22) 4 2/4/16 Cells and Organelles in Tissue Engineering • The cell is the basic unit of life • EukaryoAc cells share a basic set of organelles that are required to accomplish the generic tasks of being a cell • Cell structure correlates with cell funcAon • Specialized cells have specialized structures that enable specialized funcAons • As organisms increase in size and complexity, cell specializaAon increases and cells are divided into funcAonal groups called Assues and organs • “Brain” = “chest”? hMp://whyﬁles.org/288stemcell10/images/allin1stem_cell2.jpg The challenge of Assue engineering is being able to re-‐create the specialized cells & their various interacAons without the beneﬁt of the program of development Don’t Forget the Structure and [email protected] of of Phosphodiglycerides – the Basis for Compartments 5 2/4/16 [email protected] Cells – Single Compartment • More about the comparison to eukaroyAc cells later [email protected] Cell – [email protected] Compartments Mitochondrion Microbody Nuclear pore complex Nuclear envelope Nucleus [email protected] Nucleolus Rough ER Ribosome Centrioles Ribosome Lysosome Endoplasmic [email protected] Smooth ER Microtubules Microﬁlaments Vesicle Golgi complex Plasma membrane Cytosol Fig. 5-‐9, p. 96 6 2/4/16 My View of Membrane-‐bounded Organelles E RE G C M PMb TGN L Px SV R ER N NE Secretory pathway Spaces Rough endoplasmic reAculum (ER) Golgi apparatus (G) Trans-‐Golgi network (TGN) Secretory vesicles (SV) Endosomes (E) Recycling Endosome (RE) Lysosomes (L) Non-‐secretory pathway Spaces Cytosol (C) Nucleus (N) Mitochondrial matrix (M) Peroxisome (Px) Ribosomes (R) Secretory pathway Membranes Endoplasmic reAculum membrane Golgi apparatus membrane Trans-‐Golgi network membrane Secretory vesicle membrane Plasma membrane (PMb) Endosome membrane Lysosome membrane Nuclear envelope (NE) – conAnuous with ER Non-‐secretory pathway Membranes Mitochondrial membranes Peroxisomal membranes Endoplasmic [email protected] (ER) • Rough ER is the starAng site for protein synthesis in the secretory pathway (ribosomes are the site of protein synthesis) • ER ﬁlls most of the cytoplasm • InterconnecAng sheets • ConAnuous with the nuclear envelope • Important role in quality control for protein synthesis • IniAal glycosylaAon of membrane proteins • Vesicles bud oﬀ and carry cargo to Golgi • Other specialized funcAons such as Ca2+ storage Fig. 5-‐14, p. 99 7 2/4/16 Golgi Apparatus • Receives products of ER • Returns membrane to the ER • Further glycosylaAon of proteins • Cis and trans faces of Golgi Vesicle from ER, about to fuse with the Golgi membrane Golgi complex Cistern
ae Internal space trans face— vesicles leave Golgi from this side for other cell [email protected] • Located near the microtubule-‐ organizing center • Sends products to trans-‐Golgi network/secretory vesicles • ConsAtuAve and regulated secreAon cis face—
vesicles from ER fuse with this side Vesicles budded from Golgi containing ﬁnished product Fig. 5-‐15, p. 100 Lysosomes • Very low pH (~5) • Low pH sAmulated hydrolyAc enzymes • Lysosomal proteins targeted to lysosomes by mannose-‐6-‐PO4 • Fuse with endosomes, phagosomes, autophagic vacuoles Lysosome containing ingested material Fig. 5-‐17, p. 101 8 2/4/16 • Contains chromosomes (DNA) packaged as chromaAn • Site of DNA synthesis • Site of RNA synthesis • Site of ribosome assembly • Nuclear envelope is a double lipid bilayer • Guarded by nuclear pores Cytoplasm Ribosomes on outer surface of nuclear envelope Outer nuclear membrane (faces cytoplasm) Nuclear pore complex Space between nuclear membranes Inner nuclear membrane (faces nucleoplasm) Nuclear envelope Nucleus Nuclear pore complex Nucleoplasm Enlarged region showing phospholipid bilayer Fig. 5-‐11, p. 97 Mitochondria • Site of ATP synthesis • Double lipid bilayer – but not like nucleus • Contains DNA and ribosomes similar to prokaryotes • Protein complement has both mitochondrial and nuclear origin • Proteins enter by inserAon through pores Intermembrane compartment (between outer and inner membranes) Cristae (folds of inner membrane) Inner mitochondrial Outer membrane mitochondrial Matrix membrane (inside both membranes) Fig. 5-‐19, p. 103 Inner Matrix Outer mitochondrial (inside both mitochondrial membrane membranes) membrane Fig. 5-‐19, p. 103 9 2/4/16 [email protected] Cells – Single Compartment • All cell funcAons must occur within this space • Already know that it is similar to mitochondria • Other similariAes (ribosomes, plasma membrane, cytoplasm) • Others are unique – ﬂagella and pili have unique structures 10...
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