L7: Responding to the cellular environment

Responding to the Cellular Environment

Learning Outcomes

Understand the importance of responding to cellular and external environments in multicellular organisms.
Explain how animals respond to low oxygen levels via Hif-1α at the cellular level and nitric oxide (NO) signaling on a wider physiological level, including the effects of sildenafil on NO signaling.
Describe how GPCR signaling in photoreceptor cells effectively responds to different light levels.
Understand how cells respond to timed events like day/light cycles via circadian rhythms and developmental timing and the mechanisms involved.

Introduction

Organisms must receive information and respond appropriately.
Cell signaling pathways allow cells to communicate about their internal and external environment and respond accordingly.
In multicellular organisms, the internal environment must be regulated (homeostasis).
Key factors affecting the cellular environment:
- Nutrient availability (e.g., insulin pathway and mTOR).
- Oxygen/CO2 levels.
- Temperature.
- Light and day/night cycles.
- Physical environment (e.g., Hippo pathway).
- Damage (physical injury, pathogens).

Responding to Elevated Temperature

The heat-shock response is induced by unfolded polypeptide chains.
Heat-shock transcription factors (HSF1 in humans) activate transcription of heat-shock genes.
Heat-shock proteins bind to and prevent aggregation of unfolded polypeptides and assist in refolding proteins into proper conformations.

Responding to Low Oxygen

In metazoans, at low oxygen levels, the transcription factor Hif-1α accumulates and induces the transcription of many genes that enable cell or organism survival.
At high oxygen levels, prolyl hydroxylase PHD2 transfers an OH group to two proline residues in Hif-1α, leading to binding of tumor-suppressor protein VHL, polyubiquitinylation by an E3 complex, and destruction by the proteasome.
HIF-1α protein synthesis is regulated by activation of the phosphatidylinositol 3-kinase (PI3K) and MAP kinase pathways.
Many plants and animals express a different family of oxygen-sensitive transcription factors that involves cysteine oxidation and post-translational addition of an arginine residue, leading to polyubiquitinylation and degradation.

Hif1α and Cancer

HIF-1α is overexpressed in human cancers due to intratumoral hypoxia and genetic alterations like gain-of-function mutations in oncogenes (e.g., ERBB2) and loss-of-function mutations in tumor-suppressor genes (e.g., VHL and PTEN).
This leads to cellular changes associated with hallmarks of cancer, including angiogenesis, metastasis, and altered energy metabolism.
HIF-1α overexpression is associated with treatment failure and increased mortality.

Nitric Oxide (NO) Signaling

NO is a gas that diffuses rapidly through membranes, acting as an intracellular and extracellular messenger.
It is a reactive free radical that reacts with species containing unpaired electrons such as oxygen, superoxide, and metal ions, with a half-life of less than 10 seconds, and is metabolized to nitrate and nitrite.
Produced in response to agents like acetylcholine, which acts via a muscarinic GPCR and downstream calcium signaling.
Causes vasodilation in vascular smooth muscle, improving oxygen delivery to tissues.
NO can diffuse from a neighboring cell or be synthesized in the cell by NO synthase in response to e.g. $Ca^{2+}$ downstream of hormonal signalling.
The NO produced activates a soluble receptor (i.e., not membrane-bound) called soluble guanylate cyclase.
This produces a second messenger called 3’5’cGMP (analogous to 3’5’cAMP).
cGMP activates its own effector molecule, a kinase called protein kinase G (PKG).
PKG phosphorylates downstream target proteins to achieve the outcome in the cell.

Pharmaceutical Intervention

Nitroglycerin is used to relieve chest pains (angina) where there is compromised blood flow in the heart.
It interacts with tissue thiols to form nitrossothiols, which spontaneously decompose to release NO.
This dilates coronary arteries and improves circulation in the heart, preventing anoxia and cellular damage.
NO/oxygen mix is used as a last-resort treatment for pulmonary hypertension in neonatal intensive care.
Sildenafil (Viagra) is used: autonomic nerves release NO causing production of cGMP and relaxation of the vascular smooth muscle cells in the erectile tissue of the penis.
Viagra inhibits cGMP phosphodiesterase, potentiating NO action by preventing cGMP breakdown and prolonging erection time.
Sildenafil is a mimic of cGMP.
Viagra was approved for erectile dysfunction in the EU and US in 1998; peak sales reached nearly $2 billion in 2008. Patents expired in 2017, and it is available OTC in the UK.
A small 2013 clinical trial revealed it as a very effective treatment for alleviating dysmenorrhea (period pain) but was abandoned due to lack of funding.

Inhibiting PDE2 to Treat Heart Failure

Phosphodiesterase 2 inhibition preferentially promotes NO/guanylyl cyclase/cGMP signaling to reverse the development of heart failure.
Cyclic guanosine-3',5'-monophosphate (cGMP) plays a key role in preserving cardiac structure and function, and therapeutically targeting CGMP in HF has shown promise in experimental models and patients. Phosphodiesterases (PDEs) metabolize and curtail the actions of CGMP (and cAMP), and increased PDE activity is thought to contribute to HF pathogenesis. Herein, we show that inhibition of one specific isoform, PDE2, enhances the salutary effects of cGMP in the context of HF, and that this beneficial action facilitates a

GPCRs in Smell and Vision

Cardiac muscarinic acetylcholine GPCR regulates a $K^+$ channel.
Light stimulation of rhodopsin GPCR closes cGMP-gated $Na^+/Ca^{2+}$ channels by regulating a cGMP pathway in retinal cells.
When the rod cell is stimulated by light, a signal is relayed from the rhodopsin molecules through the cytosol to cation channels in the plasma membrane of the outer segment.
These cation channels close in response to the cytosolic signal, producing a change in the membrane potential of the rod cell.
This alters the rate of neurotransmitter release from the synaptic region of the cell. Released neurotransmitters act on retinal nerve cells that pass the signal on to the brain.

Signal Transduction Pathway in Rod Cells

In dark-adapted cells, high levels of cGMP keep the cation channels open.
In response to light, the activated Gαt subunit of the G-protein activates a phosphodiesterase that breaks down cGMP, resulting in channel closure, transient hyperpolarization of the membrane, and a reduction in neurotransmitter release.
Signal termination occurs in ~0.2 seconds, which is essential for the temporal resolution of vision.
A GAP stimulates breakdown of GTP to GDP, switching off Gαt.
Rhodopsin is also phosphorylated and binds arrestin.

Signal Amplification

When rod photoreceptors are adapted for dim light, signal amplification is enormous.
Humans can detect a flash of as few as five photons.
Each activated rhodopsin can activate 500 Gαt proteins, which activate 500 PDEs, and each PDE (active for fraction of a second) hydrolyzes hundreds of cGMPs.
Total effect: closure of thousands of cation channels causes a significant change in membrane potential.

Light Adaptation

Rod cells adapt to varying levels of ambient light by intracellular trafficking of arrestin and transducin.
In the dark (vision most sensitive to very low light levels), transducin is mostly localized in outer segment membranes, and arrestin is mostly localized in the inner segment region of the cell.
In bright light (vision is relatively insensitive to small changes in light), transducin is moved from the outer segment to the inner segment, and arrestin is moved from the inner segment to the outer segment.
Contributes to the ability to perceive images over a 100,000-fold range of ambient light levels.
Protein movement mechanism may involve microtubules and motor proteins.

Circadian Clocks

All organisms exhibit ~24-hour circadian rhythms driven by molecular clocks within cells, reset by external cues like light and dark.
All circadian clocks use negative feedback loops to control gene expression.
A negative feedback loop in which clock genes are regulated by their own protein products is conserved from flies to humans.
This feedback loop consists of two transcriptional repressors, PERIOD and TIMELESS, two transcriptional activators, CLOCK and CYCLE, several kinases and E3 ubiquitin ligases, and a light-sensitive protein (CRY in Drosophila).

Operation of the Circadian Clock in Drosophila

At dusk, concentrations of PER and TIM peak: TIM binds to PER and protects it from DBT phosphorylation and subsequent degradation.
During the night, the TIM-PER dimer is transported into the nucleus, where it binds to CLK and CYC, blocking their ability to stimulate transcription of the PER and TIM mRNAs throughout the night.
At dawn, light triggers a conformational change in the CRY-photosensitive protein, which then binds to TIM, releasing it from PER and from CLK and CYC.
During the day, CLK and CYC dimerize to stimulate the transcription of PER and TIM, but TIM and PER proteins both undergo regulated degradation.

Cyanobacteria Circadian Clock

The circadian clock in the cyanobacteria Synechococcus elongatus consists of three proteins: KaiA, KaiB, and KaiC.
At dawn, KaiC exists in a de-phosphorylated state: after the binding of KaiA, KaiC kinase activity is activated, and it autophosphorylates itself at two residues by noon.
One of the residues in KaiC slowly becomes de-phosphorylated, leading to the binding of the KaiB protein and release of KaiA.
KaiB binding to KaiC inhibits its kinase activity, leading to the full de-phosphorylated state of KaiC and release of KaiB.

The SCN is the Master Clock in Mammals

Light is detected by cells in the retina and relayed via a neuronal pathway to the suprachiasmatic nucleus (SCN) in the brain.
The SCN sends signals to other neuronal centers and glands to induce signals that regulate molecular clocks throughout the body.
One of these signals is to the pineal gland, which releases melatonin, a hormone that modulates the onset of sleep.
Individual neurons isolated from the SCN show 24-hour oscillating rhythms in action-potential firing that persist for as long as the neurons are maintained in culture.

Developmental Timing

A single gene, coding for a transcription regulator that inhibits its own expression, can behave as an oscillator.
For oscillation to occur, there must be a delay (or delays) in the feedback circuit, and the lifetimes of the mRNA and protein (which contribute to the delay) must be short compared with the total delay, which determines the period of oscillation.
It is thought that a feedback circuit like this, based on a pair of redundantly acting genes called Her1 and Her7 in the zebrafish, is the pacemaker of the segmentation clock governing somite formation.

Vertebrate Somite Formation

The temporal oscillation of gene expression in the presomitic mesoderm becomes converted into a spatial alternating pattern of gene expression in the formed somites.
In the posterior part of the presomitic mesoderm, each cell oscillates with a cycle time of 90 minutes.
As cells mature and emerge from the presomitic region, their oscillation is gradually slowed down and finally brought to a halt, leaving them in a state that depends on the phase of the cycle they happen to be in at the critical moment.
In this way, a temporal oscillation of gene expression traces out an alternating spatial pattern.
Hes7 in the mouse is the pacemaker of the segmentation clock (equivalent to Her1 and Her7 in the zebrafish).

Cell Intrinsic Timing Mechanisms

Timed expression of neural genes drives laminar organization of the cerebral cortex (mammals).
Neurons and glial cells originate from progenitor cells; successive generations of neurons migrate outwards along glial cell processes and settle sequentially along the neuroepithelium.
Neural cell types are specified by the temporal sequence of TF expression in their progenitors, so that birth order specifies both the fate and location of differentiated neurons.

Key Points

Organisms must respond to the internal and external environment appropriately, necessitating complicated signaling and homeostatic mechanisms.
In metazoans, at low oxygen levels, the transcription factor Hif-1α accumulates and induces the transcription of many genes that enable the cell or organism to survive this stress; this is often pathologically upregulated in tumors.
NO is a gas that acts as a highly diffusible intracellular and extracellular messenger (paracrine signaling) that activates soluble guanylate cyclase, which produces cGMP, which in turn activates protein kinase G (PKG).
In vascular endothelial cells, NO diffuses to the vascular smooth muscle to cause relaxation and vasodilation.
Nitroglycerin (produces NO) and sildenafil (inhibits phosphodiesterase) are pharmacological agents that target this pathway.
Rod cells in the retina use a rhodopsin GPCR to respond to light by activating the Gαt subunit of the G-protein, which in turn activates a phosphodiesterase that breaks down cGMP, resulting in cation channel closure, transient hyperpolarization of the membrane, and a reduction in neurotransmitter release, altering signaling to the brain.
Rod cells adapt to varying levels of ambient light by intracellular trafficking of arrestin and transducin.
All organisms exhibit ~24-hour circadian rhythms driven by molecular clocks within cells that use negative feedback loops to control gene expression and that can be reset by external cues like light and dark.
This feedback loop consists of two transcriptional repressors, PERIOD and TIMELESS, two transcriptional activators, CLOCK and CYCLE, several kinases and E3 ubiquitin ligases, and a light-sensitive protein.
A single gene, coding for a transcription regulator that inhibits its own expression, can behave as an oscillator in developmental patterning events, converting oscillations of gene expression into spatial patterns.