D103 MT Review

• Cells differ in morphology and function.

• Cells vary in size from about 1 µm (bacterium) to about 200 µm (oocyte).

• Organelles are only present in eukaryotic cells and are =tightly regulated in number and organization.

• Organelle dysregulation can lead to diseases.

• Many considerations (costs, time, ability to observe, complexity, etc.) go into the selection of the right model system for a scientific question.

• Microscopy (resolving distance limit = 1/2 wavelength)


• Lipids are amphipathic molecules that assemble in an energetically favorable way (bilayer, micelle, liposome)

• Lipid bilayers contain a hydrophobic core (about 3-5 nm thick), are fluid and impermeable to charged molecules.

• A biological membrane is asymmetric in respect to lipid and protein composition. Bilayer asymmetry is important for cell function.

• Selective membrane permeability is mediated by transmembrane proteins which associate with the membrane as integral, peripheral or lipid-anchored proteins.

• About 20-25 amino acids with hydrophobic character are necessary to span a membrane of 3-5 nm

• A hydrophobicity plot is a theoretical prediction of hydrophobic domains within a protein that may function as transmembrane domains


Molecules are transported across membrane by numerous transmembrane proteins

Carriers/transporters (GLUT1, GLUT2)

• highly specific for transported molecule, saturable

• undergo reversible conformational change during the transport

• active and passive transports are possible

Pumps/ATPases (Na+/K+ pump)

• energy from ATP hydrolysis is converted into mechanical work to make ion transported against their electrochemical gradients

Ion channels (gated sodium channel)

• opening is regulated (gated)

• only involved in passive transport (down gradient)

• saturable transport

• selective for size and charge of ion

Directions of transport

• Transport down the electrochemical gradient is passive and does not require energy

• Transport against the electrochemical gradient requires energy

  • “ride along” (sym- and antiport) taking advantage of the potential energy of other co-transported molecules.

  • ATP hydrolysis (Na+/K+ pump)

  • light-driven


Oxidative phosphorylation

• Electrons move down the electron transport chain, leading to the establishment of an electrochemical proton gradient used to generate ATP through ATP synthase

• ATP synthesis requires a proton impermeable inner mito membrane

• ATP synthase converts the chemical energy of the proton gradient into mechanical energy to obtain ATP (chemical energy)

Mitochondria cell biology

• mitos contain 2 functionally distinct membranes with unique protein/lipid composition and function (porins in outer membrane, cardiolipin in inner membrane)

• mitos contain their own DNA encoding for 37 genes

  • replicated, transcribed and translated in mitochondria

  • most mito activities depend on protein and RNA import from the cytosol

• mito organization adapts to a cellular energy requirement in respect to their number

• mito inheritance: maternal, mitotic segregation leads to cells with different mtDNA content

Mitochondrial diseases

• hereditary diseases caused by mutations in mitochondrial DNA and/or nuclear DNA encoding for proteins that function in mitochondria

• their severity depends on the percentage of mitochondria with mutated DNA

Immunofluorescence

• Cells are chemically fixed and stained with primary antibodies that recognize the antigen of interest and secondary antibodies that are coupled to fluorophores and can recognize the primary antibody


• The cytoskeletons support cell shape and movement

• 3 main cytoskeletal structures (actin, tubulin and intermediate filament)

• Filament actin (F-actin): assembly from ATP-bound G-actin to polarized filament

• Nucleation is the rate-limiting step involving nucleation factors

• Treadmilling occurs when cytosolic concentration of G-actin-ATP is higher than critical concentration at the (+) end and lower than the critical concentration at the (-) end (Cc+ < C cyto < Cc-)

• The cytosolic concentration of G-actin-ATP is tightly regulated

• Actin binding proteins regulate the dynamics of actin filaments

• Visualize the protein movement by using fluorescent tag (e.g., GFP, RFP)


The organization and dynamics of actin cytoskeleton is regulated by actin-binding proteins, which include capping proteins, regulators of G-actin availability and nucleators.

Cell migration:

• Depends on enhanced actin nucleation at the leading edge of the cell, dynamic attachment of cells to the substratum via focal adhesions and myosin-dependent contraction at the trailing edge.

• External signals lead to the activation of small GTPases (Rho, Rac and Cdc42), which induce changes in actin organization and then promotes cell movement

• The activity of small GTPases is determined by interacting proteins: GEFs activate the small GTPase, GAPs inactivate the small GTPase

• Dominant active (mimic GTP-bound) vs. dominant negative (mimic GDP-bound)

Skeletal muscle contraction:

• Relaxed state: F-actin and myosin filaments are prevented from interacting through specific actin binding proteins that bind actin in the same site as myosin

• During contraction: increased Ca2+ induces the binding between F-actin and myosin. If ATP is available, there is the movement of myosin towards the +end of F-actin.

Smooth muscle contraction:

• Ca2+ in the cytosol binds calmodulin → activates myosin light chain kinase.

• Active myosin light chain kinase phosphorylates myosin light chain → myosin binds actin → muscle contraction


MT formation

• heterodimer composed of a and b-tubulin as basic building block

• GTP on b-tubulin is hydrolyzed during filament formation

GTP cap:

• GTP hydrolysis on b-tubulin occurs with a slight delay after subunit addition

• High concentration of a/b-tubulin heterodimer: GTP cap forms and MT growth,

• Lack of GTP cap at low a/b-tubulin heterodimer concentration: filaments depolymerizes

Dynamic instability of microtubules

• Depend on the cytosolic concentration of GTP-bound a/b-tubulin heterodimer and the presence/absence of the GTP cap

MT nucleation at the centrosome

• Their (-) ends are capped and protected from depolymerization. Because of (-) end capping mechanism, treadmilling is less important for MTs

MTs in organelle positioning

• Depends on MT motor proteins (Kinesin – moves towards the (+) end, dynein – moves towards (-) end)

• Heavy chains of these motor proteins are responsible for ATP and MT binding


MTs during the cell cycle

• Dramatic reorganization during cell division (centrosome-nucleated radial array in interphase vs mitotic spindle → blocking MT rearrangement is a powerful tool to block cell division → anti-cancer drug, such as taxol

Centrosome number is tightly controlled during cell cycle

• Multiple centrosomes will lead to uneven chromosome separation and genomic instability

• Centrosome abnormality is a hallmark of cancer

Intermediate filaments

• Non-polar structures that provide cells with mechanical support

• They are dynamic → assembly occurs spontaneously, but energy is required for disassembly

• The nuclear envelope is supported by an intermediate filament meshwork (nuclear lamina) which disassembles during mitosis. Cell cycle-dependent reorganization of the nuclear lamina is controlled by lamin phosphorylation

• Mutations in intermediate filaments, such as lamins, lead to severe human diseases.


Adhesion proteins: Transmembrane proteins that participate in adhesion via an extracellular domain and that associate with the cytoskeleton via their intracellular domain

Example 1: Cadherins (Ca2+ binding proteins)

Ca2+-dependent conformational change, which promotes cell interaction with neighboring cells

• Adherens junctions (linkage of actin cytoskeleton)

• Desmosomes (linkage of intermediate filament)

Example 2: Integrins (heterodimer)

• Help motile cells to adhere to ECM - link between actin cytoskeleton/IF and the ECM

• Inside-out and outside-in signaling

Extracellular matrix: components are secreted by fibroblast inside the ECM

Collagen: most abundant one; involved in many diseases

Fibronectin and laminin


Principles of protein sorting

• All cellular proteins are synthesized by the same pool of cytosolic ribosomes.

• The final destination of a protein is mostly determined by a sorting signal in its primary protein sequence. The sorting signal will be recognized by a receptor that will direct it to a translocation machinery for transport into the destinated location. A protein without sorting information remains in the cytosol.

• A sorting signal for a specific compartment is generally necessary and sufficient for its delivery to this location.

• Sorting signals vary in size, charge and position within the primary sequence of a protein.

Import into the nucleus

• Proteins are fully folded during import (import occurs posttranslationally)

• The small GTPase Ran gives nuclear import directionality (localization of GAP and GEF)

• Sorting signal is not cleaved

Import into mitochondria

• Protein is kept unfolded by chaperone (Hsp70) – this requires ATP

• Several membrane complexes in the outer and inner mito membrane form translocation channels through which proteins are imported

• Membrane potential across inner mito membrane promotes protein import into matrix

• Mitochondrial matrix chaperones help the newly arrived protein to fold in the mitochondria

• Matrix sorting signal is cleaved off after import


Two different types of the ER

Smooth ER:

•Contact sites with other organelles; lipid/cholesterol synthesis

Rough ER

•Site of protein import and establishment of protein topology; mostly co-translationally

ER is an organelle of protein folding and quality control

•N-glycosylation: occur upon co-translational transfer of a precursor protein at a consensus sequence of the protein (Asn-X-Ser/Thr)

•Folding: ER chaperones help the protein to fold (BiP)

•Topology: Spatial location of positive charged amino acids determines the topology of ER transmembrane proteins

•Quality control: Protein folding in the ER is under a stringent quality control

  • If protein is not folded properly: protein leaves ER by retro-translocation, covalently modified with poly-ubiquitin, which directs protein for degradation in the proteasome (in the cytosol) - ERAD

  • If protein is folded normally: sorting into transport vesicles for Golgi delivery


Vesicle formation: (vesicle types: COPI, COPII, clathrin)

• Vesicle is derived from the donor compartment

• Initiated by recruitment and activation of small GTPase

• Leads to the recruitment of coat protein from the cytosol

• The function of coats is to recruit cargo into the vesicle and to induce membrane curvature

• The vesicle loses its coats once the GTP on the small GTPase is hydrolyzed

• Vesicle uncoating is a prerequisite for vesicle fusion (e.g., release v-SNARE)

Vesicle fusion: target membrane selection involves the following proteins:

• Tethering proteins (Rab effectors) work with Rab small GTPases to bring membranes into proximity

• SNAREs (specific SNARE pairs between vesicle and target membrane - select fusion)

• SNARE complex is disassembled by ATPase NSF and recycled for new round of transport

The secretory pathway:

• All transport steps are mediated by vesicles

• No direct connection between ER, Golgi, lysosome, plasma membrane

• Topology does not change as proteins are transported through the secretory pathway


Golgi membranes are organized as a series of functionally distinct compartments

Protein sorting in the TGN:

Resident in the Golgi, being transported to the lysosome or under secretion (regulated vs constitutive)

Clathrin – coated vesicles are responsible for transporting proteins:

From Golgi to lysosome; From the cell surface to endosome (endocytosis)

Lysosomes are acidic organelles with degradative enzymes

Hydrolases are responsible for breakdown of macromolecules

Proteins (enzymes) destined for the lysosomal compartment have M6P (phosphorylated sugar) signal. M6P receptor binds the clathrin adaptor, leading to clathrin-dependent transport of the hydrolase to the endosome. The interaction between M6P and its receptor is pH sensitive and is disrupted at the low pH of the endosome. M6P receptor is recycled while the hydrolases are delivered to the lysosome

Endocytosis transport materials to be digested by the lysosome

Endocytosed vesicles mature from early endosomes to late endosomes to lysosomes (maturation characterized by increasing acidic pH). Formation of multivesicular vesicles (MVB)

Endocytosis can concentrate particular substances (e.g., LDL cholesterol) into the cell via receptor-mediated endocytosis

Endocytosis is clathrin mediated and requires specific internalization signals which are recognized by clathrin adaptors. The interaction of LDL and its receptor is pH-regulated. Upon release of LDL from its receptor in the endosome, the receptor is recycled to back to the cell surface whereas LDL is transported to the lysosome where cholesterol is released


Protein can have different modifications that determine its diverse cellular functions

Post-translational modification (PTM)

Ubiquitination (reversible)

  • The proteasome

    • Protein degradation machinery in the cytosol

    • ATP-dependent unfolding of proteins before degradation

    • Proteins are targeted to proteasome by poly-ubiquitination

  • Ubiquitination

    • 3-step process to activate this small protein

    • Added to lysine

    • Substrates are selectively modified by E3 ligase

    • Can be reversed by de-ubiquitination enzymes (DUBs)

Phosphorylation (reversible)

  • Transient addition of PO4- to protein by kinase

  • Removed by phosphatase

  • Regulate protein conformation, interaction, localization, …

Glycosylation (N and O)

There are many other types of PTMs

Such as acetylation, methylation, sumoylation, neddylation, lipidation