D. Fundamentals of Cell Movement

______D. Fundamentals of Cell Movement

What cell types move? ______• Prokaryotes must find food, evade toxins • Free‐living ciliar and flagellar eukaryotes ______• Plants don’t have motile cells but can demonstrate both rapid and slow movements due to cell activity • Animals have both ciliar and flagellar cells ______• We also have cells that crawl, rather than swim Many cells during development and growth White blood cells responding to infection Wound healing cells ______• Muscular movements in animals result from individual cell movements ______

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______What strategies do cells employ to move? ______• Swimming through liquids: oars and propellers • Crawling on solid surfaces: grab‐pull‐release ______• Selectively contracting some cells but not others: some use motor proteins, others water volume ______• Even ‐ growing more cells, or letting some die, to move the entire structure closer or farther away! ______

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______Many prokaryotic cells have a structure composed of a membrane‐bound motor complex driving propeller‐ ______like movement of the extracellular flagellum

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The flagellum is composed ______of the helical protein flagellin ______Figure 1-18a Molecular Biology of the Cell, Fifth Edition (© Garland Science 2008)

______The helical structure of flagellin allows for two kinds of ______movement: coordinated linear vs. stationary ‘tumbling’ ______RECEPTOR CHEMOTAXIS

senses correct direction: will swim in a straight line for a ______longer time before tumbling

senses wrong direction: will tumble sooner and try a new direction at random. ______

finds the location with the highest concentration of an attractant (lowest of repellent ) ______Even at high concentrations, can distinguish very small differences in concentration. ______

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______Two levels of regulation: 1. Signal transductio to motor ______2. Control of receptor activation

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______Figure 15-73 Molecular Biology of the Cell (© Garland Science 2008) ______Ciliar and Flagellar Eukaryotes ______• The Basic Mechanism – Complex microtubular structures extend out from ______the cell body under the plasma membrane – They extend out from basal bodies rather than centrisomes ______– Immobilized dynein pulls retrograde and bends the microtubule ______– Relaxation or a counter pull creates waving ______

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Free‐living eukaryote Didiniumhas two fringes of cilium used for swimming ______

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Here it is phagocytosing another eukaryotic cell as prey ______Figure 1-32 Molecular Biology of the Cell, Fifth Edition (© Garland Science 2008) ______

Airway Epithelium ______

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______‐ G.I. epithelium ‐ Fallopian tubes ‐ Epidydimus ______

______Flagellar Animal Cells ______

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______The structure of microtubules in both cilia and flagella are the classic 9+2: ______An external ring of 9 doublets around 2 full microtubules ______

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______Figure 16-81a Molecular Biology of the Cell (© Garland Science 2008) ______

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Basal body structure is a ring of nine (9) triplets ______

Microtubules are nucleated from γ‐tubulin ______and are capped and stabilized long‐term.

Same as the centriole ______Figure 16-84a Molecular Biology of the Cell (© Garland Science 2008) ______They work as a unit by being held together with ‘radial spoke’ and ‘nexin’ proteins. As dyneins attached to one doublet attempt to walk on the adjacent one they all bend. ______

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______Figure 16-81b Molecular Biology of the Cell (© Garland Science 2008) ______

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______Figure 16-83b Molecular Biology of the Cell (© Garland Science 2008) ______

______Mechanisms of Waving ______• In long flagellum, sequential peristaltic contractions cause a whip‐ ______like back and forth motion ______• In short cilia, alternating side‐to‐side contractions or simple relaxations ______cause waving

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______Cell Migration or “Crawling” ______• The Basic Mechanism ______– Triggered by signals from outside the cell – Actin‐myosin based movement – Requires attachments to outside to pull against ______– Gotta’ drag all of the cell contents along for the ride ______

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In embryo development and ______wound healing, epithelial cells can migrate as sheets.

In general, these types of migrations are combinations ______of cell division and directed migration. ______

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______Figure 16-20 Molecular Biology of the Cell (© Garland Science 2008) ______

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Chemotaxis ______

Circumferential receptors ______Rho‐family GTPases (monomeric)

Rho‐dependent kinases ______

1. Actin monomer nucleotide exchange 2. Actin fiber polymerization and disassembly ______3. Myosin motor ATPase activity ______

______Cell type‐specific migration receptor ______Rho family monomeric GTPase Rho‐dependent kinase ______

______Cell capable of migration ______

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Circumferential distribution of migration‐inducing signaling cascades ______

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Source of signal ______

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______Leading edge extension is driven by actin polymerization.

Cell membrane is physically pushed forward by actin 1. Core of all structures is very dense actin network ______2. Completely exclude membrane enclosed organelles.

Leading edge contains everything needed for migration. 1. a cut piece without organelles will continue to migrate ______

actin growth blocked ______

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actin fibers ______

______Actin treadmilling – actin is an ATPase g-actin adds to the f-actin chain as ATP-actin ______Comes off as ADP-actin.

Rate of hydrolysis controls rate of treadmilling ______

ADP

ADP ATP ______

ADP ATP ______cofilin ATP ATP ATP ATP ATP ATP ATP profilin

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______Figure 16-90 Molecular Biology of the Cell (© Garland Science 2008) ______

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______Ultimately, the length of the f‐actin remains constant but it moves forward

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______Figure 16-87a Molecular Biology of the Cell (© Garland Science 2008) ______Attachment: Microfilament Connections Depend on Migratory Surface

All the usual suspects: focal adhesions, adherens junctions ______ATP-actin can bind to f-actin chain and to anchor proteins

Internal binding of cadherins/integrins allows external binding ______ADP-actin loses binding to f-actin and anchor protein

ADP ATP ______Detachment is as important as attachment for movement! ATP ATPATPATP ATP ATP ATP ______

______Direction of travel ______

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Migrating cells tend to follow ECM and/or cell tracts towards their target. ______

The original integrins and/or cadherins on the cell surface determine these tracts. ______

eg. Fibronectin and cadherins outside of the cell. ______What you could bind to when stationary is what you can bind to when migratory (until you change gene expression) ______

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Traction: Movement of the Cell Body Across Attached Leading Edge ______The actin structure performs a scaffolding function. Myosin pulls against actin bound to extracellular components. Myosins transport many cellular components directly. ______

cellular components ______

ADP ATP myosin motors ______

ATP ATPATPATP ATP ATP ATP ______

______Direction of travel ______One of the principal cargos of myosins that are involved in migration are intermediate filaments and microfilaments

‐ nearly everything is already bound to them! ______

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______IF (blue) ______

______Figure 16-87c Molecular Biology of the Cell (© Garland Science 2008) ______

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cell 1 ______Myosin pulls so hard that it realigns the ECM proteins. This sets up “game trails” ______wherein the first cell blazes a trail that is easier for the next cell to follow.

The later cells reach the ______destination faster than those that went ahead. ______cell 2 ______Figure 16-96 Molecular Biology of the Cell (© Garland Science 2008) ______

______Realignment of microtubules in the direction of travel allows streaming of mitochondria into the leading edge ______

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Every g‐actin placed into f‐actin and every myosin powerstroke requires a fresh ATP! ______Figure 16-87c Molecular Biology of the Cell (© Garland Science 2008) ______The special case of extravasation ______• Circulating WBC must get out of the vessel • Combines activation of the WBC with ‘Cell Rolling’, ‘Adhesion’ and ‘Diapedesis’ ______1. The presence of environmental cues associated with injury and infection change endothelial surface selectins 2. These catch closely matched WBC surface oligosaccharides ______and make them roll to a stop on endothelial surface 3. The white blood cell then activates an integrin that binds tightly to ICAM on endothelial cells ______4. Diapedesis uses basic migratory mechanisms along with WBC shape change to squeeze between endothelium ______

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______Intercellular Diapedesis ______

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______Transcellular ______Diapedesis ______

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______Molecular shortening of the sarcomere shortens the cell because the filaments attach to the plasma membrane ______

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______Each of the cells, or fibers, is attached by ECM to the other cells in its fiber bundle, or fasicle, and pulls on them ______

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______Few to many muscle fasicles make up a muscle, such as the quadriceps, all joined by connective tissue. When cells contract the force is transfered directly ______to these extracellular structures ______

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______Transference of that force through the tendon to the bone produces motion ______

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______Nastic movements are non‐directional responses to stimuli. The movement can be due to changes in turgor and the rate or frequency of these responses increases as intensity of the stimulus increases. ______

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Thignonasty/seismonasty: response to touch ______Photonasty: response to light

Nyctinasty: movements at night or in the dark ______Chemonasty: response to chemicals or nutrients

Hydronasty: response to water ______Thermonasty: response to temperature

Geonasty/gravinasty: response to gravity ______

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Tropic movements are growth or turning in response to an environmental ______stimulus that is dependent on the direction of the stimulus. Tropisms may be either positive (towards) or negative (away from) the stimulus. ______Gravitropism: response to gravity

Chemotropism: response to chemicals

Heliotropism: response to sunlight ______

Hydrotropism: response to water Phototropism: response to lights or colors ______Thermotropism: response to temperature Electrotropism: response to an electric field ______Thigmotropism: response to touch or contact Host tropism: response to pathogens ______

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______The Endomembrane System and Intracellular Trafficking ______What does a cell do when its mitochondria or lysosomes wear out? ______How does it keep the lysosomal enzymes from digesting everything in the process? ______How does a cell change the receptors on its plasma membrane when necessary? ______How does a cell duplicate EVERYTHING when it’s time to divide? ______

______Fig. 7‐3 ______Remember what the cytosol and membranes are made up of.... ______

______Phospholipid bilayer ______

Hydrophobic regions Hydrophilic of protein regions of protein ______

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Fig. 7‐9ac ______And.... ______Signaling molecule

Enzymes Receptor ______

______ATP Signal transduction (a) Transport (b) Enzymatic activity (c) Signal transduction ______

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Fig. 7‐9df ______And.... ______

______Glyco‐ protein ______(d) Cell‐cell recognition (e) Intercellular joining (f) Attachment to the cytoskeleton and extracellular Carbohydrates often play important roles on matrix (ECM) ______the plasma membrane. Covalently bonded to lipids (forming glycolipids) or more commonly to proteins (forming glycoproteins) ______

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And.... ______

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______Figure 10-41a Molecular Biology of the Cell (© Garland Science 2008) ______

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______How does the cell get all of these hydrophobic molecules to their appropriate locations – ______

Right through the middle of an aqueous ______environment?! ______

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______The endomembrane system allows containment and movement of hydrophobic and dangerous materials ______• Components of the endomembrane system: – Nuclear envelope – Endoplasmic reticulum – Golgi apparatus ______– Lysosomes – Peroxisomes – Vacuoles ______– Plasma membrane

• These are bridged by membrane vesicles ______• This process is called vesicular transport ______Copyright © 2008 Pearson Education, Inc., publishing as Pearson Benjamin Cummings ______Fig. 6‐16‐3 ______Nucleus ______Rough ER

Smooth ER ______cis Golgi ______

1. Movement from the ______nucleus Plasma outward trans Golgi membrane ______

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Fig. 6‐14 ______

Nucleus 1 µm Vesicle containing 1 µm two damaged organelles ______

Mitochondrion fragment ______Peroxisome fragment Lysosome Digestive enzymes Lysosome Lysosome ______Plasma Peroxisome membrane Digestion

Food vacuole Mitochondrion Digestion Vesicle ______(a) Phagocytosis (b) Autophagy

2. Movement from the 3. Movement within plasma membrane inward the cytosol ______

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______Figure 13-3b Molecular Biology of the Cell (© Garland Science 2008)

______Like everything else in the cell this starts in the nucleus‐ER complex.... ______The Nucleus delivers all RNAs (r,t,m) to ER for translation. a. Nuclear pores need be very large, 8 protein subunits. ______b. The outer membrane is continuous with the ER and may even have ribosomes on the nucleus proper.

c. Free ribosomes direct cytosolic translation. ______

1. Free and bound ribosomes are structurally identical

2. mRNA sequence directs them on or off the RER surface ______

3. “Free” is a relative term –they are anchored to the cytoskeleton ______

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Fig. 5‐26‐3 DNA ______

1 Synthesis of mRNA in the ______nucleus mRNA

NUCLEUS ______CYTOPLASM

mRNA 2 Movement of ______mRNA into cytoplasm Ribosome via nuclear pore

3 Synthesis ______of protein

Amino Polypeptide acids ______

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Fig. 6‐11 ______Cytosol Endoplasmic reticulum (ER) ______

Free ribosomes

Bound ribosomes ______

Large ______subunit

Small ______0.5 µm subunit TEM showing ER and ribosomes Diagram of a ribosome ______

______What kinds of things are made on free ribosomes? ______• Intermediate Filaments, Actin, Tubulin • Myosin, Kinesin, Dynein ______• Microfilament associated proteins • Microtubular associated proteins ______• 2nd messengers for signaling cascades • Glycolysis enzymes ______• Lots and lots of others....

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______What kinds are made on bound ribosomes? ______• Signal Receptors, Transporters, Channels • Cadherins, Integrins ______• Anchor protein complexes • Enzymes inside organelles ______• Secreted proteins • Lots and lots of others.... ______

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______Figure 13-3b Molecular Biology of the Cell (© Garland Science 2008) ______

Remember: Principle Functions of the ER ______1. Lipid biosynthesis ‐ phospholipids, steroids, lipoproteins (HDL, LDL) ‐ there is great cell‐specificity in lipid enzymes ______

2. Membrane-bound translation of mRNA ______- Amino‐terminus leader sequences direct placement into proper orientation ______3. Initial integration of lipids and proteins - Lipids associate based on amino acid structures ______

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______What info is in the Amino Terminus? ______• If you are a transmembrane protein, the amino terminus will get you put in the transmembrane position in the ER

• If you are a luminal or secreted protein, the amino terminus ______will get you put into the lumen of the ER

• If you are a meshwork protein, the amino terminus will get ______you attached to the ER cytosolic face

• The information is interpreted by ribosomes and/or ______chaperonins ______

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Fig. 7‐8 ______

N‐terminus NON‐CYTOSOLIC SIDE The amino ______terminus comes off the ribosome ______first and is thus the first through the ER ______membrane

C‐terminus ______CYTOSOLIC SIDE ______

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The Cytosolic v. Non‐Cytosolic ______relationship is always maintained in the endomembrane system: ______

______Once non‐cytosolic, always non‐cytosolic, etc. ______

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The Proteins that are Placed into the ER Membrane ______Determine the Lipids that Assemble from the Mix ______

Different amino acid side chains ______have binding affinity for different ER lipids ______

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Fig. 6‐16‐3 ______Nucleus ______

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Golgi ______Apparatus ______Vesicles Plasma membrane ______

______Budding, Transport , Targeting and Fusion ______1. Vesicles bud off of a membrane due to coat proteins in the membrane meshwork ______2. Vesicles are transported to their next membrane due to vesicle motor‐binding proteins ______3. Vesicles are specifically targeted to their next membrane due to vesicle membrane proteins ______4. Vesicles fuse to their destination membrane due to their lipid constituents ______

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______Figure 13-2 Molecular Biology of the Cell (© Garland Science 2008)

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______Budding, Transport and Targeting are dependent on a combination of the transmembrane and meshwork proteins of the source membrane ______

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______Figure 10-41a Molecular Biology of the Cell (© Garland Science 2008)

______The three major families of coat proteins... ______

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______Figure 13-5 Molecular Biology of the Cell (© Garland Science 2008) ______

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______Figure 13-8 Molecular Biology of the Cell (© Garland Science 2008) ______

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______Figure 13-7a, b Molecular Biology of the Cell (© Garland Science 2008) ______

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______Figure 13-7c, d Molecular Biology of the Cell (© Garland Science 2008) ______

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______Figure 13-12a Molecular Biology of the Cell (© Garland Science 2008) ______

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______Figure 13-12b Molecular Biology of the Cell (© Garland Science 2008) ______Transportation and targeting of vesicles ______

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______Transportation and targeting of vesicles ______

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______Transportation and targeting of vesicles ______

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______Fusion of two membranes depends on a reasonably good match between the ______lipids that they have assembled in the two sheaths of their bilayers. ______

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Fig. 7‐10 ER ______1 Membranes have Transmembrane distinct inside and glycoproteins outside faces Secretory ______protein The asymmetrical Glycolipid distribution of proteins, lipids, Golgi 2 apparatus and ______carbohydrates is Vesicle determined when the membrane is built by the ER ______and Golgi 3 apparatus Plasma membrane: Cytoplasmic face 4 Extracellular face ______Transmembrane Secreted glycoprotein protein Membrane glycolipid ______

______Lipid and protein signatures

The lipids and proteins exposed on the cytosolic face of a ______membrane are different from those on the non‐cytosolic face. Enough to easily tell them apart. ______

______• Lipid signature is relatively assymetric • Protein signature is absolutely assymetric • Sugars are also absolutely assymetric ______

Fig. 7‐5c ______Phospholipids Cholesterol Cerebrosides Sphingolipids ______

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______Cholesterol ______

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Fig. 7‐8 ______

Amino N‐terminus NON‐CYTOSOLIC SIDE acid ______sequences exposed on the cytosolic ______face are absolutely different from ______those on the non‐ cytosolic face C‐terminus ______CYTOSOLIC SIDE ______

______The Role of the Golgi Apparatus ______• Process vesicular proteins and lipids through covalent modifications ______

• As the vesicle progresses from cis to trans it undergoes a distinctive series of changes ______

• When the product leaves the GA it has new ______transport, targeting and fusion information ______

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______Different cis domains receive ______distinct ER products ______

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______Vesicles fuse and bud as they travel along the cisterna ______

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______Many GA alterations but none more important than sugar assembly and glycosylation. ______

100’s of human enzymes give great cell‐ ______specificity in glycosylation patterns ______

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______Plasma Membrane Targeting, Transport and Fusion ______• Constituitive Delivery and Secretion – Automatic transport from trans‐GA to PM ______– Occurs continuously as needed

• Regulated Secretion ______– Specialized vesicles dock under PM – Wait for a secondary signal to cause fusion ______

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______Lysosome Targeting, Transport and Fusion ______• Enzymes are potentially deadly to the cell ______• Optimal pH of enzyme activity is ~5.0, while the rest of the cell is maintained at ~7.2 ______• They are made in the ER and placed in the lumen, where they are inactive ______

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______Lysosome Targeting, Transport and Fusion ______• Co‐expressed with the standards: MBC, KBC, GA rab, lysosome rab ______• Also with a mannose‐6‐phosphate receptor protein –sugar activated lysosome targeting ______

• Also with an inactive hydrogen ion pump – ______lipid fusion at lysosome ONLY activates it! ______

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______The Endosomal System ______• Inward flow of vesicular material ______• Phagocytosis and pinocytosis

• Receptor removal and recycling ______

• ECM turnover and remodeling ______

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______Figure 13-1 Molecular Biology of the Cell (© Garland Science 2008) ______Fig. 7‐20c RECEPTOR‐MEDIATED ENDOCYTOSIS ______Coat protein Receptor Coated vesicle ______

Coated pit Ligand ______

A coated pit Coat and a coated protein vesicle formed ______during receptor‐ mediated endocytosis (TEMs) ______

Plasma membrane 0.25 µm ______

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______Mito Targeting, Transport and Fusion ______• Remember: The Endosymbiotic Theory ______• Their own DNA, divide by binary fission ______• Two membranes – The outer membrane is 50:50 protein:lipid ______– The inner membrane is 80:20 protein:lipid ______

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The lipids and most proteins of the inner ______membrane arise from expression of genes maintained in the mitochondrion itself ______Those of the outer membrane arise from expression of genes maintained in the ______nucleus and trafficked via ER and GA ______What of the rate limiting step enzymes? ______

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______Expressed on free ribosomes from nuclear mRNAs ______TOM COMPLEX ______

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Neurotransmitter Release ______

______Let’s put it all together.... ______

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Neurons and muscle cells in animals and phloem cells ______in plants rely on electrical signaling.

• Electricity is the energy created by the movement of charged particles ______–it’s named for the example of electrons

• When a cell uses electricity it does it by allowing ions that it has concentrated by active transport to rush from one side of the ______membrane to the other through channel proteins

• The opening and closing of the channels determines when the electrical current is flowing ______

• Voltage is a measure of how many ions are on the move

• Membrane potential is a measure of how many ions have been ______actively concentrated across a membrane ______Copyright © 2008 Pearson Education, Inc., publishing as Pearson Benjamin Cummings ______

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• Concentrated ions diffuse faster than uncharged ______molecules ______• Two combined forces, collectively called the electrochemical gradient, drive the diffusion of ions across a membrane: ______

– A chemical force (the ion’s concentration gradient) ______– An electrical force (the effect of the membrane potential on the ion’s movement) ______Copyright © 2008 Pearson Education, Inc., publishing as Pearson Benjamin Cummings ______

• An electrogenic pump is a transport protein that generates ______voltage across a membrane

• The sodium‐potassium pump is the major electrogenic ______pump of animal cells

• The main electrogenic pump of plants, fungi, and bacteria is ______a proton pump

• Mitochondria and chloroplasts use a proton pump to help ______make ATP

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______• Build the axon electrical gradient • Build the synapse ______• Build and locate the neurotransmitter vesicles • Depolarize and repolarize the plasmamembrnae ______• Transduce the signal that causes vesicular fusion

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