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What are the proposed reasons for bacteria to target proteins to specific regions of the cell, i.e. to localize their proteins?
- Bacteria cells are very large when compared to the size of the proteins.
- 1. Proteins that function together need to find each other…bacteria are huge, and proteins are TINY, so need to get in touch, and cooperate w each other sometimes
- 2. Chromosome organization and replication. Ori at the cell pole..replication results in ori separated at two poles so each daughter receives chromosome
- 3. Signaling purposes; detect the direction of the signal source and regulate response…ie chemotaxis proteins localized near flagella
- 4. Regulation of timing and site of enzyme activity, cytokinesis.
2. What mechanisms have been described for protein localization in bacteria?
- 1. Exclusion: nucleoid acts as barrier
- 2. Localized Degradation: Similiarity to developmental gradients in eukaryote embryogenesis. ie Cdr2 localized protein acts to degrade Pom1, only near the Cdr2 region.
- 3. Diffusion and capture: Diffusion in 3D (cytoplasm) or 2D (membrane). Binding with other proteins occurs after diffused…and they get localized by another protein.
- Also..geometry may be part of the answer. Internal surface of vegetative cell is composed entirely of NEG curved membrane surfaces. In a sporulating cell developing spore surrounded by membrane with POS curvature.
3a. In the Ramamurthi & Losick paper covered in class, evidence was presented indicating that membrane curvature can serve a cue for protein localization. Provide the big questions and conclusions from this paper.
- Paper Big Picture: What helps in regulation of the LOCALIZATION of cell division protein DivIVA?
- Conclusions: curvature of the cell, both concave and convex, has a role in aiding in in protein localization. Protein structure interact with phosphate heads of phospholipids
3b. Ramamurthi Methods/Results
- 1. created divIVA genes with GFP tag, both WT and with deletions.
- 151-164 last 14aa from C-terminus: no effect on localization
- 126-164 last 25aa from C-terminus: little effect on amount of polar and septal localization
- 2-50 residues from N-terminus: ALL localization LOST
- 2. Used bacteria with SirA gene (no chromosome) to show if no nucleus removed: localization persists
- 3. Used micZ (FtsZ inhibitor, blocking cytokinesis) to block septa formation: showed that without septa formation, increase in localization at the poles (usually vice versa)
- 4. Used Lysozyme to remove cell wall and create spherical shape: showed that protein needs the level of curvature to localize
4a. The Werner et. al. paper covered in class reported on a genome-wide screen for localized proteins. What organism was the focus of this study, what were the basic methods used?
- Focus of study: How common is localized protein in bacteria, using Caulobacter organism.
- Created many ORFs linked via recombinases (to transform with mCherry). Confirmed via PCR, then transferred to destination vector where expression is driven by xylose inducible promoter and ORF fused to mCherry at N or C terminus (using gXRN or gXRC genes).
- mCherry used because it will fluoresce under MANY cytoplasmic conditions
- These transformed to E. coli and then recombination induced, followed by Transfer to Caulobacter via CONJUGATION. These transformants were grown on Kan and screened for localization of the fusion protein.
4b. The Werner et. al. paper covered in class reported on a genome-wide screen for localized proteins. What organism was the focus of this study, what were the overall findings of the study?
- about 3000 ORFs were amplified and found, via high throughput methods, 300 localized proteins (12% of Caulobacter)
- 10 types of localized proteins found: pole, bipolar, stalked, midband, line, etc.
- Rate of false positives: Low, of 29 previously shown to localize, 24 were reconfirmed
- Inclusion bodies: some localization pattern/clumping may just be aggregates of protein
- Proteins that function together localize togther?: Not necessarily, when looking at CckA and CtrA, linked by ChpT. ChpT (histadine phosphotransferase) does not localize at same spot as these, although it helps w their fxn.
- Localization high at poles, near 30-40% of cell length, which is the ACD site of Caulobacter
5. What is the relationship between cilia, flagella (eukaryotic), and axonemes? What is the role of the basal body in these “organelles”? How do these organelles generate movement?
- cilia and flagella have the SAME basic structure and both play a role in cell motility. The difference is in the motion they produce. Cilia makes a stroke motion (freestyle swimming stroke) while Flagella uses a flagella motion (like a wave)
- AXONEME: the functional core of both the cilia and flagella. Axoneme is composed of MTs that are templated off of the basal body of the cell (aka the centrosome), specifically the 9 outer doublet MTs
- Movement generation: by a DYNEIN reaching out to the ADJACENT doublet and walking towards the negative end of the MT towards the basal body
6. What is the primary cilium, how does it relate to a “normal” cilium, and what role does it play in cell function?
- Primary cilium: Do Not have the central pair of microtubules that are found in normal cilia (aka: 9 + 0 rule). They are also NON-motile cilium
- Role in cell function: NOT cell motility, but functions with sensory organs, by acting as an antenna for the cell, able to send and receive signals from outside the cells
- ie Nematode olfactory neurons: odor receptors in their primary cilia
- ie Rod and cone photoreceptors: modified cilia with basal body and vestigal axoneme
- ie Epithelial and renal tubules: primary cilia present to act as sensors for Ca signaling cascade
- ie Vertebrae embryos: primary cilia in ventral node required for assymetric location of heart and liver on one side of body
7. What is intraflagellar transport (IFT) and how was it discovered? Also discuss the importance of IFT and the primary cilium.
- IFT is the transport of materials up and down the length of the flagella, under the surface of the plasma membrane, btw membrane and the microtubule doublets.
- 1st observed: timelapse video of Clamydomonas flagella, first thought as mechanism to get components from axoneme to tip of flagella
- Mechanism of axoneme assembly/turnover: Axonemes grow at their tips, so need kinesin to bring componenents to tip and dynein to bring components from tip back to cytoplasm
- IFT and primary cilium: Needed because IFT is needed for primary cilium cell signaling processes, needed to get signals up and down the axoneme of primary cilia to help with cell functions.
8. What is a ciliopathy? Provide at least 2 specific examples of ciliopathies and provide a description of the cellular processes that are affected in these ciliopathies.
- Ciliopathy: new class of diseases, resulting from a genetic disorder in cilia, anchoring structure and /or function.
- Example 1: Kartegener's syndrome: IMMOTILE cilia syndrome (ICS), defect in the action of the cilia lining the respiratory tract (causing respitory problems), fallopian tube and also of the flagella of sperm in males (causing infertility) and situs inversus.
- Autosomal recessive
- Cell process affected: Dynein in axoneme mutated, so ALL cilia becomes NON-motile…
- Except for: Primary cilia on Node are MOTILE in this case of ICS.
- Sonic hedgehog (Shh) expression leads to Nodal expression and Pitx2 to LEFT SIDE structures, vs Activin expression leading to blocking of Nodal and Snail leading to RIGHT SIDE structures.
- Example 2: Polycystic kidney disease (PKD): Late onset, causes of RENAL failure in adults
- Autosomal dominant
- Cell process affected: mutations in PKD1 and PKD2 genes, which encode polycystin 1 or polycystin 2, cause PRIMARY Cilia are NON-motile. These cilia usually detect flow in epithelial of renal (kidney) to transmit Ca signals to cells
- Hypothesis is that FLUID flow causes bend in cilia, making it OPEN Ca+ channels, causing signaling cascade to lead to cell proliferation (Cyst development)
9a. At least four distinct types of cellular processes have been associated with cilia assembly and function, what are they?
- 1 Intraflagellar transport: IFT proteins localize to cilia to aid in cilia function, such as in transporting photosensory proteins to connecting cilium.
- 2 Sensory structure/functions: cilia involved in Odorant Reception via olfactory sensory neurons. Each neuron express only 1 of 900 odorant receptors ( G-protein coupled receptors). odorant signaling proteins (ie GPCR) localized to the primary cilium
- 3 Signaling:
- a. Ion/Ca channel: Primary cilia role in signaling seen in Polycystic Kidney disease, with defective primary cilia causing Ca channel opening to lead to signaling cascade and cell proliferation.
- b Wnt, HH: ie w HH, leading to transcription activation: cilia play role in IFT with signals, such as Smo, which travels via IFT to convert GliR to GliA (active), which travels back down to nucleus to activate transcription.
- 4 Cell division: Primary cilia play a role in cell division, with their base being the centriole/centrosome
- 5 Cell motility: Mutations that cause non-motile cilium seen with Immotile cilia syndrome (Kartageners)
9b. Below is a list of defects/diseases resulting from abnormal cilia assembly or function. For each, indicate which of the cellular processes from the first part of this question is/are associated with each of the defects/diseases, and provide a brief explanation of the connection between cilia assembly or function and the mechanisms/symptoms of the disease. To put that another way, what is the mechanistic connection between cilia assembly or function and the specific class of diseases?
- Cystic diseases: with cilia involved in SENSORY organs. In Polycystic kidney disease, primary cilia are NON-motile, possibly due to a bending of the cilia causing opening of Ca channels and then signaling cascade leading to cell proliferation and cysts in liver.
- Retinitis pigmentosa: with cilia involved in SENSORY organs, and possibly involved with IFT. Mutations in kinesin KIF3A leads to accumulation of opsin and membranes at the base of the cilium causing retinal degeneration. Defect in rhodopsin blocks transport to the outer segment. Not clear if the primary cause for retinal degeneration is intraflagellar transport.
- Anosmia: cilia involved in SENSORY organs. Disease causes inability to smell, due to defective non-motile 9+0 sensory cilia present on olfactory neurons, Also a symptom of Bardt-Biedl Syndrome
- Cancer: with cilia involved in CELL DIVISION. Mutations causing loss of cilia signaling ability can causing cell proliferation and loss of cell cycle control.
- Situs inversus: with cilia and cell motility. In this case, there is mutation that interfere with normal assembly of nodal cilia. Primary cilia on node are immotile. Usually these are motile (disobey the 9+0 immotile rule). Nodal gene expression is critical for formation of either R or L side.
- Impaired wound healing: with cilia cell signaling and movement. Fibroblast primary cilia play a role in signaling to monitor directional movement of fibroblasts during wound healing
- Diabetes: with cilia sensory structure/function. It is a symptom of Bardt-Biedl Syndrome
- Skeletal patterning: with cilia involved in sensory organs. Plays a role in chemosensation and mechano sensation.