Defective kinesin heavy chain behavior in mouse kinesin light chain mutants

There is no obvious difference in the amount of KLC2 that coprecipitated with KIF5A or KIF5B between wild-type, heterozygous, and homozygous KLC1 mutant mice.

“There was no obvious difference in the amount of KLC2 that coprecipitated with KIF5A or KIF5B between wild-type, heterozygous, and homozygous KLC1 mutant mice; normal KLC1 and the KLC1 fragment were not detected in homozygous mutant immunoprecipitations.”

A very small amount of truncated KLC1 protein product can be detected in KLC1 mutant brain.

“A very small amount of the truncated KLC1 protein product could also be detected.”

In wild-type brain, all KIF5A sedimented in fractions containing KLC1 and KLC2.

“Quantitative Western blotting of sucrose gradient fractions of mutant and wild-type brain extracts revealed that in wild-type, all KIF5A sedimented in fractions containing KLC1 and KLC2.”

KLC1 mutant mice have a pool of KIF5A not associated with any known KLC subunit.

“Biochemical analyses demonstrated that KLC1 mutant mice have a pool of KIF5A not associated with any known KLC subunit.”

• Also stated in: § Abundance of KLC and KHC in KLC1 Mutants · hedged

  “The lack of any measurable change in the amounts of KIF5A and KLC2 in KLC1 mutants suggests that there is a pool of KIF5A that is not associated with any KLC in KLC1 mutants; this supposition is confirmed by the sucrose gradient analysis described below.”

• Also stated in: § Interactions of KIF5A and KIF5B with KLC in Wild-type and KLC1 Mutants · plain

  “Quantitative Western blotting of sucrose gradient fractions of mutant and wild-type brain extracts revealed that in wild-type, all KIF5A sedimented in fractions containing KLC1 and KLC2. The KLC1 mutant, however, showed a consistent shift in sedimentation of KIF5A to lighter fractions with significantly less KLC present.”

In KLC1 mutants, KIF5A colocalizes aberrantly with the peripheral cis-Golgi marker giantin in sensory and motor neuron cell bodies.

“Immunofluorescence studies of sensory and motor neuron cell bodies in KLC1 mutants revealed that KIF5A colocalized aberrantly with the peripheral cis-Golgi marker giantin in mutant cells.”

• Also stated in: § Alterations in Distribution of KIF5A, KIF5B, and COPI in Cell Bodies of the Motor and Sensory Neurons · plain

  “Interestingly, the aberrant KIF5A staining in the motor neuron cell bodies of the ventral horn was seen to colocalize only with the cis-Golgi marker giantin in KLC1 homozygous mice and not with other markers tested (Fig. 4 A).”

• Also stated in: § Alterations in Distribution of KIF5A, KIF5B, and COPI in Cell Bodies of the Motor and Sensory Neurons · plain

  “Similar colocalization of KIF5A and giantin was seen in sensory neuron cell bodies of the DRG (Fig. 4 B).”

KLC1 mutant mice are significantly smaller than their wild-type or heterozygous littermates.

“Removal of functional KLC1 resulted in significantly smaller mutant mice that also exhibited pronounced motor disabilities.”

• Also stated in: § General Observations of Mutant Mice · plain

  “These mice are, however, significantly smaller than their wild-type or heterozygous littermates (Fig. 2A and Fig. C).”

• Also stated in: § General Observations of Mutant Mice · plain

  “The size difference between wild-type or heterozygous and homozygous mice was also noticeable in adults (Fig. 2 A).”

Striking changes and aberrant colocalization were observed in the intracellular distribution of KIF5B and β′-COP in KLC1 mutants.

“Striking changes and aberrant colocalization were also observed in the intracellular distribution of KIF5B and β′-COP, a component of COP1 coatomer.”

The observed Mendelian ratio of genotypes from heterozygous matings is 1:2:1 (wild-type:heterozygous:homozygous).

“In a mixed 129/BL6 background, the predicted Mendelian ratio of 1:2:1 was observed of wild-type (KLC1 +/+; n = 25/98), heterozygous mutant (KLC1 +/−; n = 54/98), and homozygous mutant (KLC1 −/−; n = 19/98) animals.”

There is no discernible difference in levels of KIF5A or KIF5B in crude cytoplasmic lysates of brain among wild-type, heterozygous, and homozygous KLC1 mutants.

“Careful quantitation using actin as an internal control standard revealed no discernible difference in the levels of either KIF5A or KIF5B in crude cytoplasmic lysates of brain from each of the three genotypes (Fig. 2 D and Table ).”

KLC2 is still capable of forming complexes with KIF5A or KIF5B in KLC1 mutant mice.

“Immunoprecipitations with anti-KIF5A and KIF5B antibodies from brain lysates indicate that KLC2 is still capable of forming complexes with either KIF5A or KIF5B in KLC1 mutant mice (Fig. 3 A).”

KLC1 mutant mice exhibit pronounced motor disabilities.

“Removal of functional KLC1 resulted in significantly smaller mutant mice that also exhibited pronounced motor disabilities.”

• Also stated in: § General Observations of Mutant Mice · plain

  “Besides the size difference, KLC1 mutant mice also exhibited visible motor defects.”

Homozygous KLC1 mutant mice are viable.

“PCR genotyping of mouse tails from offspring of matings of heterozygous mice showed that homozygous KLC1 mutant mice are viable (Fig. 1 C).”

The KLC1 fragment is virtually absent from the cell bodies of homozygous KLC1 mutant sensory and motor neurons.

“the KLC1 fragment was virtually absent from the cell bodies.”

Western blots of brain tissue from mutant animals suggest only a very small amount of the KLC1 fragment relative to wild-type KLC1 is present.

“First, Western blots of brain tissue from mutant animals suggest that only a very small amount of the KLC1 fragment relative to wild-type KLC1 is present.”

Truncated KLC1 protein levels are dramatically reduced in the cell bodies compared with wild-type levels.

“The combined Western and staining data thus suggest that the truncated KLC1 protein levels are dramatically reduced in the cell bodies, compared with wild-type levels of KLC1.”

Sciatic nerve ligation experiments indicate KIF5A and KIF5B are able to move in a plus end directed fashion in KLC1 mutant mice.

“Ligation experiments indicate that KIF5A and KIF5B are able to move in a plus end directed fashion in KLC1 mutant mice (Fig. 6).”

In homozygous KLC1 mutant sensory and motor neuron cell bodies, normal KLC1 staining is absent.

“the strong and diffuse KLC1 staining ordinarily seen in wild-type was not observed in the cell bodies of homozygous mutant sensory (Fig. 5 C) and motor neurons (data not shown), confirming that normal KLC1 was gone”

Immunoprecipitation experiments from whole brain provide no evidence for association of the KLC1 fragment with KIF5A or KIF5B.

“Third, immunoprecipitation experiments from whole brain provide no evidence for the association of the KLC1 fragment with KIF5A or KIF5B.”

In KLC1 mutant homozygotes, KIF5A accumulates abnormally in tubular structures in motor neuron cell bodies and sensory neuron cell bodies in DRG.

“In KLC1 mutant homozygotes, KIF5A was seen to accumulate abnormally in tubular structures in both the motor neuron cell bodies (Fig. 4 A) and the sensory neuron cell bodies in the DRG (Fig. 4 B).”

KLC1 mutant mice did not exhibit neuronal problems as severe as those seen in Drosophila.

“they did not exhibit neuronal problems as severe as those seen in Drosophila.”

Double labeling experiments indicated that accumulations of KIF5A in sensory and motor neuron cell bodies colocalized with the cis Golgi marker giantin.

“Double labeling experiments indicated that accumulations of KIF5A in sensory and motor neuron cell bodies colocalized with the cis Golgi marker giantin.”

In heterozygous KLC1 mutant mice, KIF5A staining in motor and sensory neuron cell bodies is primarily diffuse, similar to wild-type.

“Mice that were heterozygous for the KLC1 mutation showed a primarily diffuse staining pattern of KIF5A in both motor and sensory neuron cell bodies (Fig. 4 C)”

Altered CM1A10 staining largely colocalizes with aberrant KIF5B staining.

“The altered CM1A10 staining largely colocalized with the aberrant KIF5B staining (Fig. 5 F).”

In heterozygous KLC1 mutant motor and sensory neurons, only faint KIF5A staining in or near the Golgi apparatus is seen, similar to wild-type.

“Similar to wild-type animals, only faint staining by KIF5A antibodies in or near the Golgi apparatus was seen in the motor and sensory neurons of heterozygous mutant animals.”

Heterozygotes are normal with respect to all phenotypes observed.

“Sixth, heterozygotes are normal with respect to all phenotypes we observed”

The punctate KIF5B distribution in homozygous KLC1 mutant sensory neurons does not colocalize with known ER, mitochondrial, or Golgi markers.

“that did not colocalize with known ER, mitochondrial, or Golgi markers (data not shown).”

We do not observe accumulation of KIF5A or KIF5B KHC chains at the cellular periphery or on filamentous elements in the cytoplasm of KLC1 mutant neurons.

“We do not observe accumulation of the KIF5A or KIF5B KHC chains at the cellular periphery or on filamentous elements in the cytoplasm of KLC1 mutant neurons.”

Sites of aberrant accumulation of KIF5A or KIF5B in DRG or spinal cord do not exhibit costaining with KLC antibodies in mutants.

“Fourth, the sites of aberrant accumulation of KIF5A or KIF5B in DRG or spinal cord do not exhibit costaining with KLC antibodies in mutants”

Immunofluorescence of sensory and motor neuron cell bodies in DRG and spinal cord reveals only faintly detectable staining of the KLC1 fragment relative to robust staining in wild-type.

“Second, immunofluorescence of sensory and motor neuron cell bodies in DRG and spinal cord reveals only faintly detectable staining of the KLC1 fragment relative to the robust staining ordinarily seen in wild-type.”

In the absence of functional KLC1, KIF5A accumulates at a site of presumed transport initiation in or near the Golgi apparatus.

“supported by our observation that in the absence of functional KLC1, KIF5A accumulates at a site of presumed transport initiation in or near the Golgi apparatus.”

KIF5B shows a similar, though less extreme, shift in sedimentation in KLC1 mutants.

“Similar, though not as extreme, behavior of KIF5B was also seen.”

In KLC1 mutant sensory neuron cell bodies, β′-COP/COPI staining is radically redistributed from a Golgi-like pattern to a more punctate pattern.

“Intriguingly, COPI distribution detected by mAb CM1A10 staining of β′-COP (Lowe and Kreis 1996) in sensory neuron cell bodies was also altered by the lack of functional KLC1.”

• Also stated in: § Alterations in Distribution of KIF5A, KIF5B, and COPI in Cell Bodies of the Motor and Sensory Neurons · plain

  “The staining pattern changed from one that resembled Golgi apparatus staining in wild-type sensory cells to a more punctate staining pattern in KLC1 mutant cells (Fig. 5 E).”

• cites Lowe and Kreis 1996Not checked yet

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KLC2 staining levels are consistently reduced in sensory neuron cell bodies of KLC1 mutant mice.

“KLC2 staining (Fig. 5 B) levels were consistently reduced in the sensory neuron cell bodies of KLC1 mutant mice.”

There are no discernible differences between accumulations in wild-type, heterozygous, and homozygous mutant KLC1 mice in sciatic nerve ligation experiments.

“There were no discernible differences between accumulations in wild-type, heterozygous, and homozygous mutant KLC1 mice.”

Neither mAb 63-90 nor polyclonal KLC1 antibodies showed costaining of the KLC1 fragment with giantin or KIF5A in homozygous KLC1 mutant cells.

“Neither mAb 63-90 or polyclonal KLC1 antibodies showed any costaining of the KLC1 fragment and giantin or KIF5A in homozygous KLC1 mutant cells.”

Sensory and motor neurons of mice lacking wild-type KLC1 have obvious alterations in intracellular localization of kinesin-I and COP-I components.

“their sensory and motor neurons have obvious alterations in the intracellular localization of kinesin-I and COP-I components.”

KLC1 is essential for proper KHC activation or targeting.

“Taken together, these data best support models that suggest that KLC1 is essential for proper KHC activation or targeting.”

Only a small amount of KIF5A or KIF5B, if any, is associated with the KLC1 fragment.

“Although the level of truncated KLC1 is greatly reduced in mutant brain, this observation suggests that only a small amount of KIF5A or KIF5B, if any, is associated with the KLC1 fragment.”

Mutants lacking KLC in Drosophila melanogaster exhibit neuronal phenotypes similar to those seen when KHC is removed.

“Mutants lacking KLC in Drosophila melanogaster (Gindhart et al. 1998) exhibit neuronal phenotypes similar to that seen when KHC is removed.”

• cites Gindhart et al. 1998 sourceNot checked yet

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Kinesin-I plays a primary role in neuronal transport.

“Previous studies with mutants lacking KHC in invertebrate organisms have all given neuronal phenotypes (Saxton et al. 1991; Gho et al. 1992; Patel et al. 1993; Hurd and Saxton 1996), suggesting that kinesin-I plays a primary role in neuronal transport.”

• cites Saxton et al. 1991 sourceNot checked yet

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• cites Gho et al. 1992 sourceNot checked yet

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• cites Hurd and Saxton 1996Not checked yet

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The KLC1 mutation is recessive.

“Heterozygous mutant mice were normal for every metric we tested, indicating that the KLC1 mutation is recessive.”

The KLC1 mutation made is functionally null with respect to the phenotypes observed, despite presence of a KLC1 fragment of low abundance.

“There are several lines of evidence that support the view that the KLC1 mutation we made is functionally null with respect to the phenotypes observed, in spite of the presence of a KLC1 fragment of low abundance.”

There appears to be a larger than normal pool of KIF5A lacking associated KLC in KLC1 mutants.

“Thus, there appears to be a larger than normal pool of KIF5A lacking associated KLC, a suggestion that was confirmed by sucrose gradient analysis.”

Both KLC and KHC mutants in Drosophila exhibit membranous clogs in the segmental nerves.

“In fact, both KLC and KHC mutants in Drosophila exhibit membranous clogs in the segmental nerves (Hurd and Saxton 1996; Gindhart et al. 1998).”

• cites Hurd and Saxton 1996Not checked yet

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• cites Gindhart et al. 1998 sourceNot checked yet

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The most plausible explanation for the cellular phenotype observed in KLC1 mutant mice is that a subset of kinesin-I holoenzyme function is disturbed.

“The most plausible explanation for the cellular phenotype observed in KLC1 mutant mice is that a subset of kinesin-I holoenzyme function is disturbed in KLC1 mutant mice.”

KIF5B is also accumulating at a presently unidentified site of transport initiation in KLC1 mutants.

“Under this view, KIF5B is also accumulating at a presently unidentified site of transport initiation in KLC1 mutants.”

The observation that faint but detectable KIF5A is seen in or near the Golgi apparatus in wild-type neurons, coupled to many previous observations of kinesin-I in association with Golgi elements in a variety of cell types, suggests that the Golgi region is a normal site of KIF5A binding.

“However, the observation that faint but detectable KIF5A is seen in or near the Golgi apparatus in wild-type neurons, coupled to many previous observations of kinesin-I in association with Golgi elements in a variety of cell types (Marks et al. 1994; Lippincott-Schwartz et al. 1995; Johnson et al. 1996), suggests that the Golgi region is a normal site of KIF5A binding.”

• cites Marks et al. 1994 sourceNot checked yet

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• cites Lippincott-Schwartz et al. 1995Not checked yet

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• cites Johnson et al. 1996Not checked yet

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The KLC1 fragment does not actively exert cellular effects.

“suggesting that the fragment does not actively exert cellular effects.”

Lethality due to null mutations of KHC and KLC in Drosophila may be a secondary phenotype due to the physiology of the organism, rather than because of a crucial requirement for certain cargoes to be transported by kinesin-I.

“Hence, lethality due to null mutations of KHC and KLC in Drosophila may be a secondary phenotype due to the physiology of the organism, rather than because of a crucial requirement for certain cargoes to be transported by kinesin-I.”

• AssumptionRests on an assumption: Drosophila axons are of much smaller caliber than mouse axons.
• AssumptionRests on an assumption: A mouse axon has more volume to accommodate membranous accumulations, allowing it to overcome blockage.

KLC1 has crucial functions even in the presence of potentially redundant KLC2 and KLC3.

“Together, these data demonstrate that KLC1 has crucial functions, even in the presence of the potentially redundant KLC2 and KLC3.”

• AssumptionRests on an assumption: KLC2 and KLC3 are potentially redundant with KLC1.

The observation that KIF5A accumulations colocalize with giantin is consistent with the hypothesis that kinesin-I plays some role in transport events involving, or initiating, at the Golgi apparatus.

“This observation is consistent with the hypothesis that kinesin-I plays some role in transport events involving, or initiating, at the Golgi apparatus.”

Giantin and mannosidase II (MannII) precisely colocalize, confirming that giantin labels the cis-Golgi compartment.

“To confirm that giantin probes were labeling cis-Golgi compartment as expected, double labeling with antibodies directed against the well characterized Golgi marker mannosidase II (MannII) were done; precise colocalization of MannII and giantin was observed (Fig. 5 A).”

The small amount of KLC1 fragment in heterozygous KLC1 mutants does not actively induce abnormal behavior of KIF5A.

“suggesting that the small amount of KLC1 fragment does not actively induce abnormal behavior of KIF5A.”

The KLC1 mutant is functionally null.

“Although a small amount of the encoded KLC1 fragment could be detected under some conditions, several lines of evidence discussed here suggest that this mutant is functionally null.”