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BIOL 404 > Article 1 > Flashcards

Article 1 Flashcards

1
Q

What is the hypothesis of this paper?

A

Myosin-1c is involved in the slow component of adaptation in hair cells

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2
Q

What is myosin-1c?

A

Myo1c is a type of myosin motor protein, in hair cells primary role is associated with adaptation, and contributes to the structural integrity of stereocilia and intracellular transport

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3
Q

What are hair cells?

A

Hair cells are the primary sensory receptor cells within the inner ear

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4
Q

What is the function of hair cells?

A

Hair cells convert mechanical energy into electrical energy that is transmitted to the central nervous system via the auditory nerve to facilitate audition

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5
Q

What is adaptation in hair cells?

A

Refers to the ability of these cells to adjust their sensitivity to continuous or sustained stimuli such as sound or head movements

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6
Q

What are the two adaptation phases?

A

Fast adaptation and slow adaptation

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7
Q

What is the slow component of adaptation in hair cells?

A
  • Completed within seconds to minutes
  • Thought to be carried out by adaptation motors
  • Motors link transduction channels to the actin core of stereocilium and adjust resting tension on the channels
  • essentially enables hair cells to adjust their sensitivity to a sustained stimulus
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8
Q

What is the fast component of adaptation in hair cells?

A
  • Is completed within milliseconds
  • Occurs when Ca2+ enters open transduction channels and binds to the site on or near the channel.
  • When Ca2+ binds, the channels close
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9
Q

What did researchers mutate in this study?

A

Tyrosine-61 of myosin-61 of myosin to glycine

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10
Q

Where is Myo1c present?

A

In bundles in sufficient quantity to carry out adaptation and is located near stereociliary tips where transduction and adaptation take place

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11
Q

What are the four lines of evidence that implicate myosin in slow adaptation?

A
  1. The adaptation motor actively generates force -> is not a passive response to stimulus
  2. Adaptation takes place within actin-rich stereocilia and myosins are the only motor molecules known to move along actin
  3. Slow adaptation requires Ca2+ and calmodulin
  4. ADP analogs and phosphate analogs (inhibitors of myosin activity) block slow adaptation
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12
Q

What is Myosin-1c thought to mediate?

A

adaptation

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13
Q

What did researchers aim to do in this study?

A

They aimed to selectively inhibit the motor activity of Myo1c to see if it had an impact on adaptation

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14
Q

What was the purpose of the chemical-genetic strategy?

A

Is used to selectively inhibit the motor activity of Myo1c with certain chemicals

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15
Q

What specific mutation was introduced into Myo1c and how did it affect its sensitivity?

A

The Y61G mutation and greatly increased its sensitivity to inhibition by N6-modified ADP analogs

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16
Q

What is the objective of creating transgenic mice expressing Y61G-Myo1c?

A

To test the impact of the Y61G mutation on Myo1c’s function in hair cells and observe its effect on the adaptation process

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16
Q

Why is the immobilization of a fraction of Myo1c considered important in the adaptation motor complex?

A

It may be sufficient to disrupt the adaptation process, which likely involves many Myo1c molecules working together

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17
Q

What was found when hair cells in transgenic mice expressing Y61G-Myo1c were exposed to N6 ADP?

A

Blocked the process of slow adaptation in hair cells

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18
Q

What was the objective in creating a mutation in Myo1c?

A

To create a mutation that sensitizes Myo1c to a specific inhibitor while having little impact on ATP hydrolysis and motor activity

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19
Q

How did the Y61G mutation affect ATP hydrolysis and motor activity in Myo1c?

A

It did not significantly affect ATP hydrolysis and motor activity; it retained velocity and kinetics similar to wild-type Myo1c

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20
Q

What is the role of tyrosine-135 in myosin II and how was it related to the Y61G mutation?

A

Forms a hydrogen bond with the N6-amino group of ADP. The Y61G mutation was designed based on this and it expanded the nucleotide-binding pocket, allowing N6-modified nucleotide analogs to bind more tightly to Y61G-Myo1c

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21
Q

Why was an ADP analog considered an appropriate inhibitor for Myo1c?

A

Because it should cause myosin to remain tightly bound to actin in a rigor-like state, effectively inhibiting its function

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22
Q

What was the effectiveness of NMB-ADP as in inhibitor of Y61G-Myo1c?

A

Was an effective and selective inhibitor of Y61G-Myo1c, allowing for precise control of Myo1c inhibition

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23
Q

How did the combination of mutant Myo1c and NMB-ADP affect in vitro motility when mixed with wild-type Myo1c?

A

Produced a dominant effect on in vitro motility. Only a small fraction of Y61G-Myo1c was required for robust inhibition by the ADP analog.

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24
Q

Why is ATP-hydrolytic activity important for efficient adaptation in the adaptation motor complex?

A

It is required for the function of all myosins in the complex to ensure efficient adaptation

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25
Q

How does the immobilization of mutant myosins by NMB-ADP affect the movement of the entire complex?

A

It halts the movement of the entire complex, effectively stopping its function

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26
Q

What approach was used to create a theoretical model for transduction and adaptation in hair cells?

A

Combined an adaptation motor formalism with adaptation motor properties inferred form in virto assays

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27
Q

According to the theoretical model, what % of Y61G-Myo1c molecules is needed to inhibit adaptation in hair cells when NMB-ADP is used?

A

10% of the total myo1c molecules

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28
Q

How did the researchers ensure that the levels and distribution of Y61G-Myo1c expression were similar to that of endogenous, wild-type Myo1c?

A

Fused approx 16kbp of mouse Myo1c genomic DNA with a cDNA encoding rat Myo1c. Aimed to match levels and cellular distribution of Y61G-Myo1c expression to that of endogenous Myo1c

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29
Q

Why was the presence of an unspliced intron between the first and second coding exons a concern in the experiments?

A

The unspliced intron between the first and second coding exons could potentially encode a functional protein, introducing uncertainty and complicating the results

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30
Q

How did the researchers address the issue of the unspliced intron?

A

Created a second transgenic construct that lacked the intron thereby ensuring that the transgene did not produce the alternative protein encoded by the unspliced intron

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31
Q

What % of the total Myo1c transcript population in the lung and utricle was accounted for by the transgene in one found line (Y61G-9018)?

A

The mRNA transcribed account for approximately 30% of the total Myo1c transcript population in the lung and 65% in the utricle

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32
Q

How did they confirm the expression of Y61G0Myo1c in transgenic hair cells?

A

Purified hair bundles from wild-type and transgenic mice and used a pan-Myo1c antibody

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33
Q

What was the % increase in total Myo1c observed in bundles from transgenic and wild-type?

A

Y61G-33 transgenic mice had 65% more total Myo1c

34
Q

What does the immunoblot data suggest about the number of Myo1c in stereocilia of Y61G-9018 transgenic mice?

A

Stereocilia of Y61G-9018 transgenic mice contain fewer than 200 molecules of Y61G-Myo1c

35
Q

Why was it challenging to selectively detect Y61G-Myo1c in bundles of transgenic hair cells using anti-epitope tag antibodies?

A

The scarcity of Myo1c made it difficult to selectively detect Y61G-Myo1c

36
Q

What approach was used to investigate whether mutant Myo1c interfered with adaptation in hair cells?

A

whole-cell, tight-seal recording and mechanical stimulation of both wild type and transgenic hair cells

37
Q

What types of adaptation dominate in mouse utricular hair cells?

A

slow adaptation

38
Q

How did researchers mechanically stimulate hair cells during the experiment?

A

by deflecting their hair bundles either with a fluid jet or with a stiff probe

39
Q

Why did researchers examine transduction and adaptation in three independent mouse lines expressing Y61G-Myo1c and in a control wild-type transgenic line?

A

Wanted to ensure that the observed effects were not due to the insertion of the transgene into a specific genomic location

40
Q

What was the purpose of analyzing transduction and adaptation in three independent mouse lines expressing Y61G-Myo1c?

A

Aimed to confirm that the observed effects on transduction and adaptation were consistent across different mouse lines expressing the Y61G mutant transgene

41
Q

Why did researchers expect that the expression of Y61G-Myo1c would have few effects on hair cell properties in the absence of the inhibitor?

A

Y61G-Myo1c behaved similarly to wild-type Myo1c in the presence of ATP in vitro

42
Q

How did the transduction and adaptation properties in hair cells from the Y61G-33 and -36 lines compared to those in wild type in the absence of NMB-ADP?

A

In the absence of NMB-ADP, the transduction and adaptation properties in hair cells from the Y61G033 and 036 lines were very similar

43
Q

What were the observed similarities in current deflection plots between wild-type and Y61G-expressing hair cells?

A

Both were very similar

44
Q

How did the resting open probability of transduction channels in hair cells expression Y61G-Myo1c compare to the wild type?

A

In the absence of stimulation, the resting open probability of transduction channels in hair cells expressing Y61G-Myo1c was higher than that for the wild-type hair cells

45
Q

What were the effects of Y61G-Myo1c expression on the rate of adaptation to negative steps in cells?

A

Was unchanged in hair cells expressing Y61G-Myo1c compared to wild-type controls

46
Q

How did the small effects of Y61G-Myo1c expression on hair cell properties in the absence of NMB-ADP align with the researchers predictions?

A

Was consistent with the researchers predictions of the effect of increased Myo1c levels in transgenic bundles

47
Q

What did the researchers do to confirm that NMB-ADP did not interfere with transduction in hair cells?

A

Examined transduction and adaptation in wild type littermates of Y61G mice and in transgenic mice expressing a wild type transgene when hair cells from these mice were dialyzed with 100 of 250 uM NMB-ADP

48
Q

What were the observed effects of dialyzing hair cells with 100 or 250 uM NMB-ADP on adaptation?

A

No effects on adaptation even after protracted dialysis of >30 min.

49
Q

How did the adaptation properties of hair cells from nontransgenic littermates compare to those from outbred CD-1 mice?

A

The adaptation properties of hair cells from nontransgenic littermates were consistent with those from outbred CD-1 mice, suggesting similar behaviour in response to stimulation

50
Q

What were the observed similarities in adaptation properties between hair cells dialyzed with NMB-ADP and those in CD-1 mice?

A

The time constant for adaptation to a positive step that opened half the channels in hair cells dialyzed with NMB-ADP was similar to that in CD-1 mice

51
Q

How did the researchers test adaptation under control conditions more thoroughly?

A

Applied an adapting step to a hair bundle and then used short test deflections to examine the effect of the adapting step on the I(X) curve

52
Q

What is the significance of a shirt in the I(X) curve during adaptation testing, and why does it occur?

A

Reflects the movement of the adaptation motor, which modulates the position of the curve

53
Q

How were the currents recorded during the adaptation testing protocol affected by prolonged dialysis with 250 uM NMB-ADP in wt-42 transgenic hair cells?

A

Did not affect how quickly and how far the I(X) curve shifted in response to positive or negative deflections

54
Q

What did researchers observe when they analyzed adaptations in Y61G transgenic hair cells with NMB-ADP in the recording pipette before the nucleotide had time to diffuse the stereocillia?

A

Observed robust transduction and adaptation in most hair cells, which was indistinguishable from recordings from other Y61G cells in the absence of the nucleotide analog

55
Q

What was the effect of delivering the ADP analog through a whole-cell pipette on adaptation in Y61G hair cells?

A

Was blocked within a few minutes when the ADP analog was delivered through a whole-cell pipette

56
Q

Why was there a delay of a few minutes in blocking adaptation when the ADP analog was delivered through a whole cell pipette

A

Was due to the time required for the analog to diffuse into the cell

57
Q

Did the block of adaptation occur in hair cells from all three mutant transgenic lines?

A

Yes

58
Q

What happened to the fraction of Y61G-Myo1c expressing hair cells that retained any adaptation to positive deflections as whole-cell dialysis proceeded?

A

The initial rate of adaptation in Y61G-myo1c hair cells was 5-fold slower than in wild type cells

59
Q

What changes were observed in the I(X) curves during adaptation testing when adaptation was inhibited by NMB-ADP in hair cells expressing Y61G-Myo1c

A

Shifted and broadened during adaptation testing when it was inhibited by NMB-ADP in hair cells expressing Y61G-Myo1c

60
Q

What were the observed changes in amplitudes of responses in Y61G hair cells as NMB-ADP blocked adaptation?

A

As NMB-ADP blocked adaptation, amplitudes of responses fell by 30-60% within a few minutes

61
Q

How did the stability of transduction currents in Y61G hair cels with NMB-ADP compared to that of the wild type?

A

Transduction currents in Y61G hair cells with NMB-ADP typically showed a significant decrease in amplitude, while those in wild-type hair cells with
NMB-ADP or Y61G hair cells without the analog remained stable over ten or more minutes

62
Q

What was the noticeable change in channel closing when a hair bundle was returned to its rest position after a positive deflection in Y61G hair cells?

A

Channel closing slowed

63
Q

What happens when large negative deflections are applied to hair bundles, and how were these responses affected by NMB-ADP in hair cells expressing Y61G-Myo1c?

A

Remove external forces from adaptation motors and permit them to climb along the actin cytoskeleton

64
Q

Did wild type hair cells dialyzed with NMB-ADP show a similar loss of negative adaptation as observed in Y61G hair cells?

A

No

65
Q

What was the fraction of Y61G-9018 hair cells that did not show adaptation at the beginning of the recording and what happened to these cells during the recording?

A

A high fraction did not show adaptaiton and most of these cells lost adaptation to both positive and negative deflections if the recording continued for 8 min

66
Q

Why did they think that fast adaptation may persist when slow adaptation is blocked?

A

Fast and slow mechanisms are distinct. In some Y61G-33 and Y61G-9018 hair cells, NMB-ADP blocked slow adaptation and revealed a residual fast component, which suggested that fast adaptation may persist when slow adaptation is blocked

67
Q

What was the time constant for the fast decay of current in the observed hair cells with a residual fast component?

A

The time constant for the fast decay of current was 4.9 ± 1.3 ms in the observed hair cells with a residual fast component.

68
Q

During which type of deflection was the fast decay component observed, and how long did it typically last in most cells?

A

The fast decay component was observed during a positive deflection and typically remained for the duration of the recording in most cells.

69
Q

Why do traditional methods like targeted gene deletion have limitations in determining the function of specific molecules in complex biological systems?

A

Traditional methods like targeted gene deletion have limitations because they may only test the first essential role for a molecule, favor developmental phenotypes that mask later physiological roles, and can be affected by redundancy and protein degradation rates.

70
Q

What was the behavior of mutant Myo1c in the absence of NMB-ADP, and why was this behavior advantageous for the study?

A

Mutant Myo1c behaved almost normally in the absence of NMB-ADP and was expressed at lower levels than wild-type Myo1c. This behavior was advantageous because it ensured normal development of hair cells prior to electrophysiological recording and allowed for acute inhibition with NMB-ADP.

71
Q

Why was it concluded that Myo1c must participate in adaptation based on the results of this study?

A

It was concluded that Myo1c must participate in adaptation because adaptation was rapidly inhibited in hair cells expressing Y61G-Myo1c only when NMB-ADP was supplied, indicating Myo1c’s involvement.

72
Q

What advantages did the specific inhibitor-sensitization strategy used in this study have over similar approaches for modifying enzymes?

A

The specific inhibitor-sensitization strategy used in this study retained nearly normal enzymatic utilization of ATP, unlike other approaches that eliminated the activity of modified enzymes with the native nucleotide.

73
Q

What was the potential drawback of using N6-modified ADP analogs as inhibitors, and how did the researchers circumvent these drawbacks in their study?

A

N6-modified ADP analogs are negatively charged and must be introduced into cells by whole-cell dialysis, microinjection, or cell permeabilization, and they can be converted into triphosphates, changing their physiological effects. The researchers circumvented these drawbacks by studying one cell at a time, controlling the cytoplasmic nucleotide concentration by dialysis, and including a high concentration of ATP in the pipette to inhibit binding of NMB-ADP molecules to adenylate kinase.

74
Q

Why would inactivation of Myo1c’s native ATPase activity be undesirable in the experiments?

A

Inactivation of Myo1c’s native ATPase activity would be undesirable in the experiments because the motor presumably requires ATP-dependent motility to move it to the site of adaptation.

75
Q

What was the key advantage of studying one cell at a time in the experiments involving N6-modified ADP analogs?

A

Studying one cell at a time allowed for precise control of the cytoplasmic nucleotide concentration and avoided metabolic conversion of the analog, ensuring the accuracy of the results.

76
Q

What alternative interpretation did the researchers consider, and why did they find it highly unlikely

A

that Y61G-Myo1c does not actively participate in adaptation but resides along the actin tracks of the adaptation-motor complex, and NMB-ADP immobilizes these myosin molecules. They found this explanation highly unlikely due to the system’s geometry and the low ratio of Y61G-Myo1c to actin in a stereocilium. Adaptation to deflections as small as 300 nm was completely blocked by NMB-ADP, and the chance disposition of Y61G-Myo1c molecules next to the adaptation motor in all stereocilia was improbable.

77
Q

What surprising effect did the researchers observe in Y61G hair cells when adaptation was blocked by NMB-ADP, and what might be the cause of this effect?

A

Transduction current amplitudes usually fell by approximately 30%–60% when adaptation was blocked by NMB-ADP. This effect might be due to the complete loss of some of the ∼50 individual transduction units in a hair cell, and it is reminiscent of a similar effect caused by calmodulin inhibitors.

78
Q

How did the inhibition of slow adaptation affect the properties of the transduction channel, and why is this effect significant?

A

Inhibition of slow adaptation affected the properties of the transduction channel by causing channels to close more slowly when bundles were returned to their rest position. This effect is significant because it suggests that the inhibition of slow adaptation indirectly influences the behavior of the transduction channel

79
Q

What limitations or uncertainties exist regarding the findings related to Myo1c’s role in adaptation in hair cells?

A

The present data are confined to the neonatal mouse vestibular system, and it remains possible that other myosin isozymes mediate adaptation in mature vestibular hair cells or in auditory hair cells

80
Q

What is the significance of the separation of slow and fast adaptation in hair cells, and why might fast adaptation be difficult to observe in control cells?

A

The significance of the separation of slow and fast adaptation in hair cells is that it allows for the independent investigation of Ca2+ and second-messenger control of fast and slow adaptation. Fast adaptation might be difficult to observe in control cells because it is most pronounced for stimuli smaller than those usually delivered, and utricular hair cells are optimized for detecting low-frequency stimuli.

81
Q

What are the implications of the finding that Myo1c participates in adaptation in hair cells?

A

The finding that Myo1c participates in adaptation in hair cells implies that this motor is a component of the hair cell transduction complex. It also suggests that Myo1c plays a role in the transduction of mechanical stimuli into electrical signals in hair cells. Additionally, this discovery may assist in identifying other components of the complex.

82
Q

How does the chemical-genetic strategy described in the paragraph benefit future research in understanding the functions of Myo1c and other essential molecules in complex biological systems?

A

The chemical-genetic strategy described in the paragraph provides a valuable tool for defining the functions of Myo1c in various cell types, as well as the functions of other myosin isozymes in multiple cell types. It can also be adapted for studying the functions of other essential molecules in complex biological systems, making it a versatile approach for future research.

BIOL 404 (20 decks)

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